JP6370058B2 - Method for producing rubusoside - Google Patents
Method for producing rubusoside Download PDFInfo
- Publication number
- JP6370058B2 JP6370058B2 JP2014033304A JP2014033304A JP6370058B2 JP 6370058 B2 JP6370058 B2 JP 6370058B2 JP 2014033304 A JP2014033304 A JP 2014033304A JP 2014033304 A JP2014033304 A JP 2014033304A JP 6370058 B2 JP6370058 B2 JP 6370058B2
- Authority
- JP
- Japan
- Prior art keywords
- lactic acid
- acid bacteria
- stevia
- stevioside
- rubusoside
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- YWPVROCHNBYFTP-OSHKXICASA-N rubusoside Chemical compound O([C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O YWPVROCHNBYFTP-OSHKXICASA-N 0.000 title claims description 62
- YWPVROCHNBYFTP-UHFFFAOYSA-N Rubusoside Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC1OC(CO)C(O)C(O)C1O YWPVROCHNBYFTP-UHFFFAOYSA-N 0.000 title claims description 55
- 238000004519 manufacturing process Methods 0.000 title claims description 33
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 192
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- 239000004310 lactic acid Substances 0.000 claims description 111
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- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 claims description 75
- 244000228451 Stevia rebaudiana Species 0.000 claims description 67
- UEDUENGHJMELGK-HYDKPPNVSA-N Stevioside Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UEDUENGHJMELGK-HYDKPPNVSA-N 0.000 claims description 47
- OHHNJQXIOPOJSC-UHFFFAOYSA-N stevioside Natural products CC1(CCCC2(C)C3(C)CCC4(CC3(CCC12C)CC4=C)OC5OC(CO)C(O)C(O)C5OC6OC(CO)C(O)C(O)C6O)C(=O)OC7OC(CO)C(O)C(O)C7O OHHNJQXIOPOJSC-UHFFFAOYSA-N 0.000 claims description 47
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 36
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
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Landscapes
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Description
本発明は、ステビア及び甜茶に含まれる甘味成分であるルブソシドの製造方法に関する。より詳細には、ステビア又はステビオシドを原料とし、乳酸菌、好ましくはラクトバチルス属菌、さらに好ましくはラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）による微生物変換を利用した、ルブソシドの製造方法に関する。 The present invention relates to a method for producing rubusoside, which is a sweetening component contained in stevia and strawberry tea. More specifically, the present invention relates to a method for producing rubusoside using stevia or stevioside as a raw material and utilizing microbial conversion by lactic acid bacteria, preferably Lactobacillus spp., More preferably Lactobacillus casei Hasegawa (accession number: FERM BP-10123). .
ステビアや甜茶に含まれる甘味成分であるルブソシドは、スクロースの１００倍の甘味成分を有するがノンカロリーであり、抗う蝕性、抗血管新生、抗アレルギー作用等を有することが報告されている甘味料である。ルブソシドは、ノンカロリーの甘味成分として、過去に注目されたが、ステビアや、甜茶の原料となる植物（甜葉懸鈎子等）等の植物からの抽出による大量生産が難しいため、商品化には至らなかった。ステビアに含有されるルブソシドの量はそれほど多くはない。また、甜茶には、ステビアと比較して、ルブソシドが豊富に含有されているが、甜茶の原料となる植物の栽培が中国の一部の地域に限られ、また天候による影響から収穫量も一定ではないとされるため、ステビアの中心甘味成分であるステビオシドやレバウディオシドＡと比較して、市場価格が数倍から１０倍とも言われている（非特許文献１及び２）。更に、ルブソシドには、抗がん剤や、その他難溶性薬剤の溶解性を高める天然可溶化剤としての働きが報告されており、その利用価値が注目されている（特許文献１）。 Rubusoside, which is a sweetening ingredient contained in stevia and strawberry tea, has a sweetening ingredient 100 times that of sucrose but is non-caloric and has been reported to have anti-cariogenic, anti-angiogenic, anti-allergic action, etc. It is. Rubusoside has been attracting attention in the past as a non-caloric sweetening ingredient, but it is difficult to mass-produce by extraction from plants such as stevia and plants that are used as raw materials for tea (such as coconut leaf coconut). There wasn't. The amount of rubusoside contained in stevia is not very large. In addition, tea tea is richer in rubusoside than stevia, but the cultivation of the plant that is the raw material for tea tea is limited to some regions in China, and the yield is constant due to the influence of the weather. Therefore, compared to stevioside and rebaudioside A, which are the central sweet components of stevia, the market price is said to be several to ten times (Non-patent Documents 1 and 2). Furthermore, rubusoside has been reported to act as a natural solubilizing agent that enhances the solubility of anticancer agents and other poorly soluble drugs, and its utility value is attracting attention (Patent Document 1).
ルブソシドの大量生産方法としては、麹菌（Aspergillus）由来の酵素を利用して、ステビオシドを原料としてルブソシドの生産を行なう手法が報告されている（非特許文献１及び２）。また、精製酵素でなく、クリセオバクテリウム（Chryseobacterium）の休止菌体による同様の変換も報告されている（非特許文献３）。しかしながら、非特許文献１及び２に記載された方法では、予め酵素を大量に精製する必要があり、その精製のため工程管理項目が増える、酵素精製のための菌体の増殖速度が遅い、麹菌には食経験上安全でないものも存在する、等といった問題があった。また、非特許文献３のクリセオバクテリウムについても、食経験上十分に安全なものではなく、病原性を有する可能性も排除できないため、安全性に問題があった。従って、食しても十分に安全であり、かつ精製酵素を使用するのではなく、菌自体を生体触媒、あるいは適宜乾燥固定した死菌粉体としてそのまま利用可能な菌を使用した、より簡便で安全な、ルブソシドの大量生産方法が求められていた。 As a mass production method of rubusoside, a method of producing rubusoside using stevioside as a raw material using an enzyme derived from Aspergillus has been reported (Non-patent Documents 1 and 2). Moreover, the same conversion by the resting cell of Chryseobacterium (Chryseobacterium) is reported instead of a purified enzyme (nonpatent literature 3). However, in the methods described in Non-Patent Documents 1 and 2, it is necessary to purify the enzyme in large quantities in advance, the number of process control items increases for the purification, the cell growth rate for enzyme purification is slow, and koji mold Have problems such as those that are not safe in eating experience. Also, the Criseobacterium of Non-Patent Document 3 is not safe enough in terms of eating experience, and the possibility of having pathogenicity cannot be excluded. Therefore, it is safe enough to eat, and instead of using purified enzymes, it is simpler and safer to use bacteria that can be used as they are as biocatalysts or as dead bacteria powders that are appropriately dried and fixed. There was also a need for a method for mass production of rubusoside.
本発明は、上記現状に鑑み、食しても十分に安全であり、安価で簡便かつ安全なルブソシドの製造方法を提供することを課題とする。 In view of the above-mentioned present situation, an object of the present invention is to provide a method for producing rubusoside that is sufficiently safe to eat, inexpensive, simple and safe.
本発明者らは、上記課題を解決するために鋭意研究を重ねた結果、驚くべきことに、ステビアを、食経験上安全である乳酸菌、好ましくはラクトバチルス属菌、さらに好ましくはラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）を用いて処理することにより、ステビア中に大量に含有されるステビオシドを基に、工業的に有利にルブソシドを生産することができることを見出した。
本発明者らは、上記以外にも下記するように種々の思いがけない新知見を得て、さらに鋭意検討を重ねて本発明を完成するに至った。
As a result of intensive studies to solve the above-mentioned problems, the present inventors have surprisingly found that Stevia is a lactic acid bacterium that is safe in food experience, preferably Lactobacillus spp., More preferably Lactobacillus casei Hasegawa. It has been found that rubusoside can be produced industrially advantageously based on stevioside contained in a large amount in stevia by treatment with a strain (accession number: FERM BP-10123).
In addition to the above, the present inventors have obtained various unexpected new findings as described below, and have further conducted intensive studies to complete the present invention.
即ち、本発明は、以下のルブソシドの製造方法等に関する。
[１]ステビアを、乳酸菌を用いて処理する工程を含むことを特徴とするルブソシドの製造方法。
[２]乳酸菌が、ラクトバチルス属の乳酸菌である前記[１]に記載の製造方法。
[３]乳酸菌が、ラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）である前記［１］又は［２］に記載の製造方法。
[４]ステビアが、ステビオシドを含有する前記[１]〜[３]のいずれか一項に記載の製造方法。
[５]乳酸菌を用いた処理温度が、２５〜６０℃である前記[１]〜[４]のいずれか一項に記載の製造方法。
[６]ステビオシドを、乳酸菌を用いて処理する工程を含むことを特徴とするルブソシドの製造方法。
[７]乳酸菌が、ラクトバチルス属の乳酸菌である前記[６]に記載の製造方法。
[８]乳酸菌が、ラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）である前記[６]又は[７]に記載の製造方法。
[９]乳酸菌を用いた処理温度が、２５〜６０℃である前記[６]〜[８]のいずれか一項に記載の製造方法。
That is, the present invention relates to the following methods for producing rubusoside.
[1] A method for producing rubusoside, comprising a step of treating stevia with lactic acid bacteria.
[2] The production method according to [1], wherein the lactic acid bacterium is a lactic acid bacterium belonging to the genus Lactobacillus.
[3] The production method according to [1] or [2], wherein the lactic acid bacterium is Lactobacillus casei Hasegawa strain (Accession Number: FERM BP-10123).
[4] The production method according to any one of [1] to [3], wherein the stevia contains stevioside.
[5] The production method according to any one of [1] to [4], wherein a treatment temperature using lactic acid bacteria is 25 to 60 ° C.
[6] A method for producing rubusoside, comprising a step of treating stevioside with lactic acid bacteria.
[7] The production method according to [6], wherein the lactic acid bacterium is a Lactobacillus lactic acid bacterium.
[8] The production method according to [6] or [7], wherein the lactic acid bacterium is Lactobacillus casei Hasegawa strain (Accession Number: FERM BP-10123).
[9] The production method according to any one of [6] to [8], wherein the treatment temperature using lactic acid bacteria is 25 to 60 ° C.
本発明によれば、食経験上安全な原料を使用して、安価で簡便かつ、食しても十分に安全なルブソシド、特に副産物としてヒトの健康に有害な物質を含まないルブソシドの大量生産方法を提供することができる。
また、本発明によれば、精製酵素ではなく、生体触媒、あるいは適宜乾燥固定した死菌粉体としてそのまま利用が可能な菌を使用することができるため、簡便なルブソシドの大量生産方法を提供することができる。
さらに、本発明によれば、ルブソシドを安価で簡便に製造することができるため、ルブソシドを甘味料や天然可溶化剤として実用化する新規かつ有用な産業を創造することができる。
According to the present invention, a method for mass production of rubusoside, which is cheap, simple and sufficiently safe to eat, particularly rubusoside which does not contain a substance harmful to human health as a by-product, using ingredients that are safe from dietary experience. Can be provided.
In addition, according to the present invention, a simple method for mass production of rubusoside can be provided because not a purified enzyme but a biocatalyst or a fungus that can be used as it is as a dead bacteria powder appropriately dried and fixed can be used. be able to.
Furthermore, according to the present invention, since rubusoside can be easily produced at low cost, a new and useful industry for practical application of rubusoside as a sweetener or natural solubilizer can be created.
以下、本発明を詳細に説明する。
本発明のルブソシドの製造方法は、ステビアを、乳酸菌を用いて処理する工程を含むことを特徴とする。
本発明のルブソシドの製造方法は、ステビアを、乳酸菌を用いて処理する工程以外の工程を含んでも良い。
Hereinafter, the present invention will be described in detail.
The method for producing rubusoside of the present invention comprises a step of treating stevia with lactic acid bacteria.
The method for producing rubusoside of the present invention may include steps other than the step of treating stevia with lactic acid bacteria.
本発明において使用されるステビアは、通常、従来公知の方法により得られるものを使用することができる。前記ステビアは、例えば、従来公知の方法により得られるものをそのまま使用することができるし、従来公知の方法により得られるものを、水洗いしただけのもの、水洗いしてさらに乾燥したもの、水洗いして蒸した後乾燥したもの、それらを任意の大きさに裁断、粉砕、又はペースト状化したものを使用できる。ステビアとしては、天然品もその加工品も支障なく本発明で使用できる。加工品としては、例えば、ステビアの乾燥物、裁断物、粉砕物、抽出物、ペースト等が挙げられる。前記乾燥方法、裁断方法、粉砕方法、抽出方法、ペースト状化方法は、従来公知の方法を使用することができる。 As the stevia used in the present invention, those obtained by a conventionally known method can be used. As for the stevia, for example, those obtained by a conventionally known method can be used as they are, and those obtained by a conventionally known method are simply washed with water, washed with water and further dried, washed with water. Steamed and dried products, or those cut into an arbitrary size, pulverized, or pasted can be used. As stevia, both natural products and processed products can be used in the present invention without any problem. Examples of processed products include dried stevia, cut products, pulverized products, extracts, pastes, and the like. Conventionally known methods can be used for the drying method, the cutting method, the pulverizing method, the extracting method, and the pasting method.
本発明において使用されるステビアの部位は、特に限定されず、どの部位でも使用することができるが、例えば、ステビアの葉等を好適に使用することができる。
また、前記ステビアは、成分としてステビオシドを含有することが好ましい。ステビア中のステビオシドの含有量は、特に限定されないが、ステビア全量に対して、好ましくは、約０．１〜２０重量％であり、より好ましくは、約１〜１５重量％である。
The portion of stevia used in the present invention is not particularly limited, and any portion can be used. For example, stevia leaves can be preferably used.
The stevia preferably contains stevioside as a component. The content of stevioside in stevia is not particularly limited, but is preferably about 0.1 to 20% by weight, more preferably about 1 to 15% by weight, based on the total amount of stevia.
本発明において使用される乳酸菌としては、特に限定されないが、例えば、ラクトバチルス属、ストレプトコックス属、エンテロコッカス属、ラクトコッカス属、ビフィズス属等の公知の乳酸菌が挙げられる。乳酸菌は、好ましくは、ラクトバチルス属、ラクトコッカス属、ビフィズス属等の乳酸菌であり、より好ましくは、ラクトバチルス属の乳酸菌等である。これらの乳酸菌は、１種単独で又は２種以上を組み合わせて使用することができる。 Although it does not specifically limit as lactic acid bacteria used in this invention, For example, well-known lactic acid bacteria, such as Lactobacillus genus, Streptococcus genus, Enterococcus genus, Lactococcus genus, Bifido genus, are mentioned. The lactic acid bacteria are preferably lactic acid bacteria such as Lactobacillus genus, Lactococcus genus, and Bifido genus, and more preferably Lactobacillus lactic acid bacteria. These lactic acid bacteria can be used individually by 1 type or in combination of 2 or more types.
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（Lactobacillus acidophilus）、ラクトバチルス・ブレビス（Lactobacillus brevis）、ラクトバチルス・ブフネリ（Lactobacillus buchneri）、ラクトバチルス・カゼイ（Lactobacillus casei）、ラクトバチルス・デルブリュッキイ（Lactobacillus delbrueckii）、ラクトバチルス・ファーメンタム（Lactobacillus fermentum）、ラクトバチルス・ヘルベティカス（Lactobacillus helveticus）、ラクトバチルス・ケフィア（Lactobacillus kefir）、ラクトバチルス・カゼイ（Lactobacillus casei）、ラクトバチルス・パラカゼイ（Lactobacillus paracasei）、ラクトバチルス・プランタラム（Lactobacillus plantarum）、ラクトバチルス・ラムノーサス（Lactobacillus rhamnosus）、ラクトバチルス・サリバリウス（Lactobacillus salivarius）等が挙げられる。これらはいずれも公知の乳酸菌であり、公知の細胞供給施設（例えばＡＴＣＣ（American Type Culture Collection））より入手可能である。 Examples of lactic acid bacteria belonging to the genus Lactobacillus include, for example, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus casei, Lactobacillus casei Brucki (Lactobacillus delbrueckii), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus kefir, Lactobacillus kefir, Lactobacillus casei, Lactobacillus caseact, Lactobacillus caseact paracasei), Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus salivarius, etc. And the like. These are all known lactic acid bacteria and are available from known cell supply facilities (for example, ATCC (American Type Culture Collection)).
前記ラクトバチルス属の乳酸菌の中でも、ルブソシドの生産能力が高い点で、ラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）が好ましい。ラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ ＢＰ−１０１２３）は、立行政法人産業技術総合研究所 特許生物寄託センター（住所：郵便番号３０５−８５６６ 日本国 茨城県つくば市東１丁目１番地１ 中央第６）に寄託されている（受託日：平成１５年（２００３年）８月１１日、受託番号：ＦＥＲＭ ＢＰ−１０１２３）。 Among the lactic acid bacteria belonging to the genus Lactobacillus, Lactobacillus casei Hasegawa strain (Accession No .: FERM BP-10123) is preferable because of its high ability to produce rubusoside. Lactobacillus casei Hasegawa strain (Accession No .: FERM BP-10123) is the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Address: Postal Code 305-8565, 1-1-1 Higashi 1-chome Tsukuba City, Ibaraki Prefecture, Japan (Deposit date: August 11, 2003, deposit number: FERM BP-10123).
本発明のルブソシドの製造方法において、ステビアを、乳酸菌を用いて処理する方法は、ステビアと乳酸菌を接触させて処理する方法であれば特に限定されず、従来公知の方法を用いることができる。乳酸菌処理は、例えば、乳酸菌を用いた培養が好ましい。乳酸菌を用いた培養方法は、従来十分に確立されていて、本発明もそれに従ってよい。
前記乳酸菌処理は、例えば、ステビアを含有する培地を常法により加熱減菌処理した後、該培地に乳酸菌を接種し、培養することで処理する方法が挙げられる。また、予めステビアを含有しない培地にて乳酸菌を増殖させ、遠心分離により菌体を得た後、該菌体に滅菌蒸留水又はＰＢＳ（−）等の滅菌リン酸緩衝生理食塩水にてステビアを抽出した混合溶液をそのまま無菌的に加えて培養することで、乳酸菌処理を行ってもよい。後の精製作業を考慮すると、余計な培地成分を含有しない滅菌水中での処理がより好ましい。なお、処理に用いる乳酸菌は生菌体のままでもよい。生菌体としては増殖期の菌体でも良いし、休止菌体を用いても良い。あるいは適宜溶媒により固定・乾燥を経た死滅粉体を用いても良いが、生菌体が好ましい。
滅菌方法としては、例えば、加熱滅菌、高圧滅菌、濾過滅菌等が挙げられ、これらに限定されることなく従来公知の方法を使用することができる。また、死滅粉体は、従来公知の方法を用いて取得することができる。
In the method for producing rubusoside of the present invention, the method of treating stevia with lactic acid bacteria is not particularly limited as long as it is a method of treating stevia with lactic acid bacteria, and conventionally known methods can be used. The lactic acid bacteria treatment is preferably, for example, culture using lactic acid bacteria. The culture method using lactic acid bacteria has been well established in the past, and the present invention may be followed accordingly.
Examples of the lactic acid bacteria treatment include a method in which a medium containing stevia is heat-sterilized by a conventional method, and then the medium is inoculated with lactic acid bacteria and cultured. In addition, lactic acid bacteria are proliferated in a medium not containing stevia in advance, and the cells are obtained by centrifugation. Then, the cells are subjected to stevia with sterile distilled water or sterile phosphate buffered saline such as PBS (−). A lactic acid bacterium treatment may be performed by adding the extracted mixed solution aseptically and culturing. In view of subsequent purification operations, treatment in sterile water that does not contain extra medium components is more preferable. In addition, the lactic acid bacteria used for a process may remain a living microbial cell. As live cells, cells in the growth phase may be used, or resting cells may be used. Alternatively, dead powder that has been appropriately fixed and dried with a solvent may be used, but viable cells are preferred.
Examples of the sterilization method include heat sterilization, high-pressure sterilization, filter sterilization, and the like, and a conventionally known method can be used without being limited thereto. Moreover, dead powder can be acquired using a conventionally well-known method.
本発明において使用される培地は、特に限定されないが、例えば、乳酸菌の培養に通常使用される炭素源、窒素源、ミネラル源等を含むもの等を使用することができ、天然培地又は合成培地等を用いることができる。好ましくは、液体培地を用いる。 The medium used in the present invention is not particularly limited. For example, a medium containing a carbon source, a nitrogen source, a mineral source, etc., which are usually used for culturing lactic acid bacteria can be used. Can be used. Preferably, a liquid medium is used.
炭素源としては、特に限定されないが、例えば、グルコース、フルクトース、ガラクトース、マンノース、ラクトース、スクロース、セロビオース、廃糖蜜、グリセロール等が挙げられ、好ましくは、グルコース、スクロース等である。窒素源としては、特に限定されないが、無機態窒素源としては、例えば、アンモニア、アンモニウム塩等が挙げられ、有機態窒素源としては、例えば、ペプトン、ポリペプトン、尿素、アミノ酸、タンパク質、大豆ぺプチド等のペプチド類等が挙げられる。窒素源は、好ましくは、ペプトン、ポリペプトン、ペプチド、アミノ酸等である。また、ミネラル源としては、特に限定されないが、酵母エキスや肉エキスの他、Ｋ、Ｐ、Ｍｇ、Ｓ等を含む、例えば、リン酸一水素カリウム、リン酸二水素カリウム、硫酸マグネシウム等が挙げられる。これらの炭素源、窒素源、ミネラル源は、１種単独で又は２種以上を組み合わせて使用することができる。
培地中の炭素源及び窒素源の濃度は、乳酸菌が生育できる通常の濃度であればよく、特に限定されない。培養開始時の炭素源濃度は、通常は、培地全量に対して約０．１〜１５重量％が好ましく、約１〜１０重量％がより好ましい。培養開始時の窒素源の濃度は、通常は、培地全量に対して約０．０５〜１０重量％が好ましく、約０．１〜５重量％がより好ましい。
Although it does not specifically limit as a carbon source, For example, glucose, fructose, galactose, mannose, lactose, sucrose, cellobiose, molasses, glycerol etc. are mentioned, Preferably, glucose, sucrose, etc. are mentioned. The nitrogen source is not particularly limited, and examples of the inorganic nitrogen source include ammonia and ammonium salts. Examples of the organic nitrogen source include peptone, polypeptone, urea, amino acid, protein, and soybean peptide. And the like. The nitrogen source is preferably peptone, polypeptone, peptide, amino acid or the like. Moreover, it is although it does not specifically limit as a mineral source, In addition to yeast extract and meat extract, K, P, Mg, S etc. are included, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate etc. are mentioned. It is done. These carbon sources, nitrogen sources, and mineral sources can be used alone or in combination of two or more.
The concentration of the carbon source and nitrogen source in the medium is not particularly limited as long as it is a normal concentration at which lactic acid bacteria can grow. The carbon source concentration at the start of culture is usually preferably about 0.1 to 15% by weight, more preferably about 1 to 10% by weight, based on the total amount of the medium. The concentration of the nitrogen source at the start of the culture is usually preferably about 0.05 to 10% by weight, more preferably about 0.1 to 5% by weight with respect to the total amount of the medium.
前記培地は、前記の窒素源、炭素源、ミネラル源に加えて、さらに、無機質、有機質、ｐＨ緩衝剤等を添加しても良い。無機質や有機質としては、特に限定されないが、例えば、硫酸アンモニウム、リン酸カリウム、塩化マグネシウム、酢酸ナトリウム、食塩、鉄、マンガン、モリブデン、各種ビタミン類等が挙げられ、これらは１種単独で又は２種以上を組合せて使用することができる。
ｐＨ緩衝剤としては、特に限定されないが、例えば、炭酸カルシウム等が好ましく挙げられる。
本発明において、ステビアは、原料として培地に添加される。ステビアは、天然物でもその加工品でも良い。加工品としては、例えば、ステビアの乾燥物、裁断物、粉砕物、抽出物、ペースト等が挙げられる。
ステビアの使用量は、特に限定されないが、培地全量に対して、好ましくは、約１〜５０重量％であり、より好ましくは、約５〜４０重量％である。
前記培地は、本発明の効果を奏する限り、上述したステビア以外の成分や添加剤を含んでも良い。
In addition to the nitrogen source, carbon source, and mineral source, the medium may further contain an inorganic substance, an organic substance, a pH buffering agent, and the like. Examples of inorganic and organic substances include, but are not limited to, ammonium sulfate, potassium phosphate, magnesium chloride, sodium acetate, sodium chloride, iron, manganese, molybdenum, various vitamins, and the like. A combination of the above can be used.
Although it does not specifically limit as a pH buffer, For example, a calcium carbonate etc. are mentioned preferably.
In the present invention, stevia is added to the medium as a raw material. Stevia can be natural or processed. Examples of processed products include dried stevia, cut products, pulverized products, extracts, pastes, and the like.
The amount of stevia used is not particularly limited, but is preferably about 1 to 50% by weight, more preferably about 5 to 40% by weight, based on the total amount of the medium.
The medium may contain components other than stevia and additives as long as the effects of the present invention are exhibited.
前記培地のｐＨは、例えば約３〜７とすることが好ましく、約６〜７とすることがより好ましい。ｐＨを制御してもよく、酸又はアルカリを用いてｐＨの調整を行うことができる。 The pH of the medium is preferably about 3 to 7, for example, and more preferably about 6 to 7. The pH may be controlled, and the pH can be adjusted using an acid or an alkali.
乳酸菌を用いた処理の好ましい実施態様として、下記のものが例示される。
予めステビアを含有しない培地に乳酸菌を接種して培養し、遠心分離により菌体を得た後、該菌体に滅菌蒸留水又はＰＢＳ（−）等の減菌リン酸緩衝生理食塩水を加えることにより、乳酸菌を含有する培地が好ましく得られる。該乳酸菌含有培地に、予め公知の方法で滅菌処理を施した前記ステビア又はその加工品を別途添加して、培養することにより、ステビアを乳酸菌を用いて処理した生産物を好ましく取得することができる。前記乳酸菌含有培地に添加するステビア又はその加工品としては、前記［００１２］に記載のステビアを使用することができるし、ステビアに抽出溶媒を添加してステビア成分を抽出させることにより得られる抽出液、ステビア及び抽出溶媒の懸濁液そのままのもの、又は該懸濁液を常法により加熱して得られる熱水抽出液を使用することもできる。前記懸濁液を加熱する温度は、特に限定されないが、通常、約４０〜９０℃である。
また、培地として、前記抽出液、懸濁液又は熱水抽出液を使用することもでき、該培地を減菌した後、該培地に乳酸菌を直接接種して培養することにより、ステビアを乳酸菌を用いて処理した生産物を好ましく取得することもできる。
これらの工程において使用されるステビアは、ステビアの葉を乾燥粉末化したもの、あるいは該粉末を適宜溶媒で抽出したものが好ましい。
The following are illustrated as a preferable embodiment of the process using lactic acid bacteria.
Inoculate and culture lactic acid bacteria in a medium that does not contain stevia in advance, obtain bacterial cells by centrifugation, and then add sterile distilled water or sterilized phosphate buffered saline such as PBS (-) to the bacterial cells Thus, a medium containing lactic acid bacteria is preferably obtained. A product obtained by treating stevia with lactic acid bacteria can be preferably obtained by separately adding the sterilized sterilized stevia or a processed product thereof to the lactic acid bacteria-containing medium in advance and culturing. . As stevia or processed product thereof added to the lactic acid bacteria-containing medium, the stevia described in [0012] can be used, and an extract obtained by adding an extraction solvent to stevia and extracting a stevia component In addition, a suspension of stevia and extraction solvent as it is, or a hot water extract obtained by heating the suspension by a conventional method can also be used. Although the temperature which heats the said suspension liquid is not specifically limited, Usually, it is about 40-90 degreeC.
Alternatively, the extract, suspension, or hot water extract can be used as a medium. After sterilizing the medium, the medium is directly inoculated with lactic acid bacteria and cultured, whereby stevia is transformed into lactic acid bacteria. It is also possible to preferably obtain the product processed by using.
The stevia used in these steps is preferably a stevia leaf obtained by dry powdering or a powder obtained by appropriately extracting the powder with a solvent.
前記抽出溶媒は、特に限定されないが、例えば、蒸留水、イオン交換水、生理食塩水、有機溶媒等が挙げられる。これらは１種単独で又は２種以上を組合せて使用することができる。前記有機溶媒としては、メタノール、エタノール、ｎ−プロパノール、イソプロパノール、ｔ−ブタノール等の低級アルコール；グリセリン、プロピレングリコール、１，３−ブチレングリコール等の液状多価アルコール；アセトン等のケトン類；酢酸エチルエステル等のエステル類等が挙げられる。これらは１種単独で又は２種以上を組合せて使用することができる。前記抽出溶媒は、好ましくは、蒸留水、エタノールと水の混合溶媒等であり、より好ましくは蒸留水である。 The extraction solvent is not particularly limited, and examples thereof include distilled water, ion exchange water, physiological saline, and organic solvents. These can be used alone or in combination of two or more. Examples of the organic solvent include lower alcohols such as methanol, ethanol, n-propanol, isopropanol, and t-butanol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; ketones such as acetone; ethyl acetate Examples include esters such as esters. These can be used alone or in combination of two or more. The extraction solvent is preferably distilled water, a mixed solvent of ethanol and water, and more preferably distilled water.
前記培地に使用されるステビアと抽出溶媒の重量比は、ステビアの種類、形状、及び乾燥状態等に応じて適宜選択され得るが、例えば、ステビア／抽出溶媒の重量比は、好ましくは、約１／１００〜５０／１００であり、より好ましくは、約５／１００〜４０／１００である。 The weight ratio of stevia and extraction solvent used in the medium can be appropriately selected according to the type, shape, and dry state of stevia. For example, the weight ratio of stevia / extraction solvent is preferably about 1 / 100 to 50/100, more preferably about 5/100 to 40/100.
本発明において、前記培地に乳酸菌を接種する条件は、特に限定されず、例えば、該培地中に乳酸菌を約０．０１〜３０％接種することが好ましく、約０．５〜１０％接種することがより好ましい。 In the present invention, the conditions for inoculating the medium with lactic acid bacteria are not particularly limited. For example, the medium is preferably inoculated with about 0.01-30% of lactic acid bacteria, and inoculated with about 0.5-10%. Is more preferable.
本発明において、ステビアを、乳酸菌を用いて処理する温度は、微生物変換が効率的に実施できれば特に限定されないが、例えば、約１５〜６５℃が好ましく、約２５〜６０℃がより好ましい。
また、乳酸菌を用いて処理する処理時間は、前記培地の組成、該培地の乳酸菌接種量、前記処理温度等に応じて適宜設定され得るが、例えば、約１２〜３３６時間が好ましく、約２４〜１６８時間がより好ましい。乳酸菌処理は、好気条件下で行ってもよく、嫌気条件下で行っても良い。
In the present invention, the temperature at which stevia is treated with lactic acid bacteria is not particularly limited as long as microbial conversion can be carried out efficiently, but is preferably about 15 to 65 ° C, and more preferably about 25 to 60 ° C.
The treatment time for treatment with lactic acid bacteria can be appropriately set according to the composition of the medium, the amount of lactic acid bacteria inoculated in the medium, the treatment temperature, etc. For example, about 12 to 336 hours are preferable, and about 24 to 168 hours is more preferred. The lactic acid bacteria treatment may be performed under an aerobic condition or an anaerobic condition.
本発明のルブソシドは、上述のようにしてステビアを乳酸菌を用いて処理して得られた生産物から、従来公知の方法を用いて、ルブソシドを採取することによって得られる。採取方法としては、ろ過、遠心分離等による粗精製又は精製、殺菌（加熱殺菌等）、希釈、濃縮、乾燥等が挙げられる。これらの採取を行なう工程を本発明の製造方法に含んでも良い。 The rubusoside of the present invention can be obtained by collecting rubusoside from a product obtained by treating stevia with lactic acid bacteria as described above using a conventionally known method. Examples of the sampling method include rough purification or purification by filtration, centrifugation, etc., sterilization (heat sterilization, etc.), dilution, concentration, drying and the like. You may include the process of collecting these in the manufacturing method of this invention.
本発明により製造されるルブソシドは、例えば、上述のようにしてステビアを乳酸菌を用いて処理して得られた生産物を、常法を用いて粗精製又は精製することにより該生産物の上澄み液を取得し、該上澄み液を希釈又は濃縮後に乾燥させることにより取得することができるし、前記生産物そのものを乾燥することにより取得することもできる。前記粗精製又は精製は、分離カラムを用いた精製、有機溶媒を用いた液相分離精製等により行なうことができる。このようにして得られる乾燥物を、従来公知の方法により濃縮、結晶化、再結晶等によってさらに精製しても良い。 The rubusoside produced according to the present invention is obtained by subjecting a product obtained by treating stevia with lactic acid bacteria as described above to a crude supernatant or purifying the product using a conventional method, for example. Can be obtained by diluting or concentrating the supernatant and drying it, or by drying the product itself. The crude purification or purification can be performed by purification using a separation column, liquid phase separation purification using an organic solvent, or the like. The dried product thus obtained may be further purified by concentration, crystallization, recrystallization and the like by a conventionally known method.
本発明は、ステビオシドを、乳酸菌を用いて処理する工程を含むルブソシドの製造方法も含有する。該製造方法は、ステビオシドを、乳酸菌を用いて処理する工程以外の工程を含んでも良い。 This invention also contains the manufacturing method of rubusoside including the process of processing stevioside using lactic acid bacteria. The production method may include steps other than the step of treating stevioside with lactic acid bacteria.
本発明において使用されるステビオシドは、通常、従来公知の方法により得られるものを使用することができる。ステビオシドは、例えば、市販のステビオシドを使用することができ、市販のステビオシド粉末をそのまま使用することができるし、市販のステビオシド粉末を溶媒に溶解させた溶解液を使用することができる。またステビアから抽出、精製したものを使用してもよい。
ステビオシドを溶解させる溶媒は、特に限定されないが、例えば、蒸留水、イオン交換水、生理食塩水、有機溶媒等が挙げられる。これらは１種単独で又は２種以上を組合せて使用することができる。前記有機溶媒としては、アセトニトリル；ジメチルスルホキシド；メタノール、エタノール、ｎ−プロパノール、イソプロパノール、ｔ−ブタノール等の低級アルコール；グリセリン、プロピレングリコール、１，３−ブチレングリコール等の液状多価アルコール；アセトン等のケトン類；酢酸エチルエステル等のエステル類等が挙げられる。これらは１種単独で又は２種以上を組合せて使用することができる。前記ステビオシドを溶解させる溶媒は、好ましくは、アセトニトリル、ジメチルスルホキシド、蒸留水、エタノール、含水エタノール等であり、より好ましくは、蒸留水、含水エタノール等である。
As the stevioside used in the present invention, those obtained by a conventionally known method can be used. As stevioside, for example, a commercially available stevioside can be used, a commercially available stevioside powder can be used as it is, and a solution obtained by dissolving a commercially available stevioside powder in a solvent can be used. Moreover, you may use what was extracted and refine | purified from stevia.
The solvent for dissolving stevioside is not particularly limited, and examples thereof include distilled water, ion exchange water, physiological saline, and organic solvents. These can be used alone or in combination of two or more. Examples of the organic solvent include acetonitrile; dimethyl sulfoxide; lower alcohols such as methanol, ethanol, n-propanol, isopropanol, and t-butanol; liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol; Ketones; esters such as ethyl acetate; These can be used alone or in combination of two or more. The solvent for dissolving the stevioside is preferably acetonitrile, dimethyl sulfoxide, distilled water, ethanol, hydrous ethanol or the like, more preferably distilled water, hydrous ethanol or the like.
ステビオシドを、乳酸菌を用いて処理する工程を含む本発明の製造方法の好ましい態様は、ステビアの代りにステビオシドを使用する以外は、上述した、ステビアを、乳酸菌を用いて処理する工程を含む本発明の製造方法と同様である。 A preferred embodiment of the production method of the present invention including a step of treating stevioside with lactic acid bacteria includes the above-described step of treating stevia with lactic acid bacteria, except that stevioside is used instead of stevia. This is the same as the manufacturing method.
上述のようにして得られたルブソシドは、甘味料として使用することができ、該ルブソシドをそのままで、食品、健康食品、サプリメント、医薬品等の製造原料として使用することができるし、該ルブソシドを常法により適宜加工したものを、食品、健康食品、サプリメント、医薬品等に使用することもできる。 The rubusoside obtained as described above can be used as a sweetener, and the rubusoside can be used as it is as a raw material for producing foods, health foods, supplements, pharmaceuticals, etc., and the rubusoside is usually used. Those appropriately processed by law can be used for foods, health foods, supplements, pharmaceuticals, and the like.
前記食品とは、経口的に摂取されるものを意味し、飲料、半固形食品、固形食品、粉末食品等が挙げられる。半固形食品、固形食品としては、例えば、ドロップ、キャンディー、チューインガム等の菓子類；クッキー、クラッカー、ビスケット、ポテトチップス、パン、ケーキ、チョコレート、ドーナツ、プリン、ゼリー等の洋菓子；煎餅、羊羹、大福、おはぎ、饅頭、カステラ等の和菓子；アイスクリーム、アイスキャンディー、シャーベット、ジェラート等の冷菓；食パン、フランスパン、クロワッサン等のパン類；うどん、そば、きしめん等の麺類；かまぼこ、魚肉ソーセージ等の魚肉練り製品；ハム、ソーセージ、ハンバーグ、コーンビーフ等の畜肉製品；塩、胡椒、みそ、しょう油、ソース、ドレッシング、マヨネーズ、ケチャップ、甘味料、辛味料等の調味類；明石焼き、たこ焼き、もんじゃ焼き、お好み焼き、焼きそば、焼きうどん等の鉄板焼き食品；チーズ、ハードタイプのヨーグルト等の乳製品；納豆、厚揚げ、豆腐、こんにゃく、団子、漬物、佃煮、餃子、シューマイ、コロッケ、サンドイッチ、ピザ、ハンバーガー、サラダ等の各種総菜；ビーフ、ポーク、チキン等の畜産物；海老、帆立、蜆、昆布等の水産物等が挙げられる。
粉末食品としては、例えば、野菜・果実類、植物、酵母、藻類等を粉末にした各種粉末；油脂類・香料類（バニラ、柑橘類、かつお等）を粉末固形化したもの等が挙げられる。
The said food means what is taken orally, and a drink, a semi-solid food, a solid food, a powdered food, etc. are mentioned. Semi-solid foods and solid foods include, for example, confectionery such as drops, candy, and chewing gum; Western confectionery such as cookies, crackers, biscuits, potato chips, bread, cakes, chocolate, donuts, pudding, jelly; Japanese confectionery such as ice cream, ice candy, sherbet and gelato; Bread such as bread, French bread and croissant; Noodles such as udon, buckwheat and kishimen; Fish meat such as kamaboko and fish sausage Kneaded products; meat products such as ham, sausage, hamburger, corn beef; seasonings such as salt, pepper, miso, soy sauce, sauce, dressing, mayonnaise, ketchup, sweetener, pungent; Akashi-yaki, takoyaki, monjayaki, okonomiyaki , Fried noodles, fried udon, etc. Teppanyaki food; dairy products such as cheese, hard-type yogurt; various prepared dishes such as natto, fried chicken, tofu, konjac, dumpling, pickles, boiled dumplings, shumai, croquettes, sandwiches, pizza, hamburgers, salads; beef, Examples include livestock products such as pork and chicken; marine products such as shrimp, scallop, salmon and kelp.
Examples of the powdered food include various powders obtained by powdering vegetables / fruits, plants, yeasts, algae and the like; oils / fragrances (vanilla, citrus fruits, bonito, etc.) powdered and solidified.
飲料としては、例えば、スープ、味噌汁等の飲食品；インスタントコーヒー、インスタント紅茶、インスタントミルク、インスタントスープ、インスタント味噌汁等の粉末飲食品；ウイスキー、バーボン、スピリッツ、リキュール、ワイン、果実酒、日本酒、中国酒、焼酎、ビール、アルコール度数１％以下のノンアルコールビール、発泡酒、酎ハイ等のアルコール飲料；果汁（例えば、リンゴ、ミカン、ブドウ、バナナ、ナシ、ウメの果汁等）入り飲料、野菜汁（例えば、トマト、ニンジン、セロリ、キュウリ、スイカの野菜汁等）入り飲料、果汁および野菜汁入り飲料、清涼飲料水、牛乳、豆乳、乳飲料、ドリンクタイプのヨーグルト、コーヒー、ココア、茶飲料（紅茶、緑茶、麦茶、玄米茶、煎茶、玉露茶、ほうじ茶、ウーロン茶、ウコン茶、プーアル茶、ルイボスティー茶、ローズ茶、キク茶、ハーブ茶（例えば、ミント茶、ジャスミン茶）等）、栄養ドリンク、スポーツ飲料、ミネラルウォーター等の非アルコール飲料等が挙げられる。 Examples of the beverage include food and drink such as soup and miso soup; powdered food and drink such as instant coffee, instant tea, instant milk, instant soup and instant miso soup; whiskey, bourbon, spirits, liqueur, wine, fruit liquor, sake, China Alcoholic beverages such as liquor, shochu, beer, non-alcohol beer with alcohol content of 1% or less, sparkling liquor, strawberry high; beverages containing fruit juice (eg, apple, mandarin, grape, banana, pear, ume juice), vegetable juice (For example, tomato, carrot, celery, cucumber, watermelon vegetable juice, etc.), beverages containing fruit juice and vegetable juice, soft drinks, milk, soy milk, milk beverages, drink-type yogurt, coffee, cocoa, tea beverages ( Black tea, green tea, barley tea, brown rice tea, sencha, gyokuro tea, houji tea, oolong tea, u Down tea, Pu'er tea, Rooibos tea, rose tea, chrysanthemum tea, herbal tea (for example, mint tea, jasmine tea), etc.), energy drinks, sports drinks, non-alcoholic beverages such as mineral water and the like.
前記健康食品とは、保健、健康維持・増進等の目的とした食品組成物を意味し、認可された特定機能性食品や、特定機能性食品の認可のないいわゆる健康食品が含まれる。前記健康食品は、例えば、液体又は半固形、固形の製品であって、クッキー、せんべい、ゼリー、ようかん、ヨーグルト、まんじゅう等の菓子類、清涼飲料、栄養飲料、スープ等が挙げられる。また、そのままお湯や水に溶かして飲用しても良い。 The health food means a food composition for the purpose of health, health maintenance / promotion, etc., and includes an approved specific functional food and a so-called health food for which no specific functional food is approved. The health food is, for example, a liquid, semi-solid or solid product, and includes confectionery such as cookies, rice crackers, jelly, yokan, yogurt and manju, soft drinks, nutritional drinks, soups and the like. Moreover, you may melt | dissolve in hot water or water as it is, and drink.
前記サプリメントとは、栄養素等を補うための栄養補助食品、栄養機能食品等を意味するだけではなく、健康の保持・回復・増進等のために役立つ機能等を有する健康補助食品、健康機能食品等をも意味する。このようなサプリメントの形状としては、例えば、タブレット状、丸状、カプセル（ハードカプセル、ソフトカプセル、マイクロカプセルを含む）状、粉末状、顆粒状、細粒状、トローチ状、液状（シロップ状、乳状、懸濁状を含む）等が挙げられる。 The supplement does not only mean nutritional supplements for supplementing nutrients, functional nutritional foods, etc., but also health supplements, functional functional foods, etc. that have functions that are useful for health maintenance, recovery, enhancement, etc. Also means. Examples of such supplements include tablets, rounds, capsules (including hard capsules, soft capsules, microcapsules), powders, granules, fine granules, troches, liquids (syrups, milks, suspensions). Including turbidity).
本発明の製造方法により得られたルブソシドを医薬品に使用する場合、例えば、経口投与用剤、非経口投与剤等の製剤に使用することができる。経口投与用剤の剤型としては、例えば、錠剤、被覆錠剤、顆粒剤、散剤、丸剤、トローチ剤、カプセル剤等の固形剤；エリキシル、シロップ、懸濁液等の液剤等の剤型が挙げられる。非経口投与剤の剤型としては、例えば、注射剤（静脈注射、動脈注射、筋肉注射、皮下注射、皮内注射、腹腔内注射、脊髄内注射、硬膜外注射、関節内注射、歯周組織内注射、歯槽骨周辺への注射）、経皮剤、経腸剤、点滴剤、外用剤、坐剤等が挙げられる。 When the rubusoside obtained by the production method of the present invention is used for a pharmaceutical product, it can be used, for example, in preparations for oral administration, parenteral administration and the like. Examples of dosage forms for oral administration include solid preparations such as tablets, coated tablets, granules, powders, pills, troches and capsules; liquid forms such as elixirs, syrups and suspensions. Can be mentioned. Examples of the dosage form of parenteral agents include injections (intravenous injection, arterial injection, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intraspinal injection, epidural injection, intraarticular injection, periodontal injection Intra-tissue injection, injection around the alveolar bone), transdermal agent, enteral agent, instillation agent, external preparation, suppository and the like.
本発明の製造方法により得られるルブソシドは、前記の食品、健康食品、サプリメント、医薬品の製造工程において、あるいは最終製品に、従来公知の方法を用いて添加することができる。これらの食品、健康食品、サプリメント、医薬品は、病的な血管新生やアレルギー作用等の予防、改善又は治療に用いることができる。また、本発明の製造方法により得られるルブソシドは、抗がん剤や、難溶解性薬剤の溶解性を高める天然可溶化剤として使用することもできる。 The rubusoside obtained by the production method of the present invention can be added in the above-mentioned food, health food, supplement, pharmaceutical production process or to the final product using a conventionally known method. These foods, health foods, supplements, and pharmaceuticals can be used for prevention, improvement, or treatment of pathological angiogenesis, allergic effects, and the like. In addition, rubusoside obtained by the production method of the present invention can also be used as a natural solubilizer that enhances the solubility of anticancer agents and poorly soluble drugs.
本発明を以下の実施例及び比較例によって具体的に説明するが、本発明はこれらによって限定されるものではない。
尚、以下の実施例及び図において、ＰＢＳ（−）とは、リン酸緩衝生理食塩水を意味し、乳酸菌とはラクトバチルス カゼイ ハセガワ菌株（受託番号：ＦＥＲＭ−ＢＰ−１０１２３）を意味する。
The present invention will be specifically described with reference to the following examples and comparative examples, but the present invention is not limited thereto.
In the following examples and figures, PBS (-) means phosphate buffered saline, and lactic acid bacteria means Lactobacillus casei Hasegawa strain (accession number: FERM-BP-10123).
（実施例１）
＜ステビオシド−乳酸菌処理サンプル及びステビオシドサンプルの調製＞
予め一般乳酸菌接種用培地（ニッスイ社製）に乳酸菌（ラクトバチルス カゼイ ハセガワ菌株）を一白金耳植え、３７℃で静置培養した後、遠心及びＰＢＳ（−）にて菌体ペレットの洗浄を行い、乳酸菌１×１０１０ｃｆｕを含むＰＢＳ（−）１ｍＬ溶液を１．５ｍＬチューブへ用意した。一方で、市販のステビオシド（長良サイエンス社製）を、予め８０％アセトニトリル溶液に２０ｍｇ／ｍＬのステビオシド濃度となるように溶解したストック溶液を用意した。該ストック溶液を、乳酸菌菌体が含まれているＰＢＳ（−）溶液へステビオシドの最終濃度が４０μｇ／ｍＬになるように加え、３７℃で４８時間静置した。これをステビオシド−乳酸菌処理サンプル１とした。また、ネガティブコントロールとして、乳酸菌を含まないＰＢＳ（−）にステビオシドを最終濃度４０μｇ／ｍＬになるよう前記ストック溶液を加え、同様に３７℃で４８時間静置したものも用意した。これをステビオシドサンプル１とした。
＜ＨＰＬＣによる分析＞
上記の通り得られたステビオシド−乳酸菌処理サンプル１及びステビオシドサンプル１を遠心し、上清１０μＬを以下の条件下でＨＰＬＣへ供した。ＨＰＬＣ（島津製作所社製）では分離カラムにＹＭＣ−Ｐａｃｋ ＯＤＳ−Ａ （Ｉ．Ｄ． ２５０×４．６ｍｍ、Ｓ−５μｍ、１２ｎｍ；ＹＭＣ社製）を用いた逆相系を使用し、移動相として水及びアセトニトリルを用いたグラジエント条件下で分析を行った。カラムオーブンを３７℃に設定したまま１０％アセトニトリル溶液から開始し、０分から２５分までに１００％まで濃度を上げ、その後１００％アセトニトリルによる洗浄工程を経る条件を用いた。流速１ｍＬ／分で２１０ｎｍでの検出を行った。
ステビオシド−乳酸菌処理サンプル１及びステビオシドサンプル１のＨＰＬＣ測定データを、それぞれ図１及び図２に示す。
Example 1
<Preparation of Stevioside-Lactic Acid Bacteria Treated Sample and Stevioside Sample>
A lactic acid bacterium (Lactobacillus casei Hasegawa strain) is preliminarily planted in a medium for inoculation of general lactic acid bacteria (manufactured by Nissui Co., Ltd.), statically cultured at 37 ° C., and then the cell pellet is washed with centrifugation and PBS (−). A 1 mL solution of PBS (−) containing 1 × 10 10 cfu of lactic acid bacteria was prepared in a 1.5 mL tube. On the other hand, a stock solution in which a commercially available stevioside (manufactured by Nagara Science Co., Ltd.) was previously dissolved in an 80% acetonitrile solution to a stevioside concentration of 20 mg / mL was prepared. The stock solution was added to a PBS (−) solution containing lactic acid bacteria so that the final concentration of stevioside was 40 μg / mL, and allowed to stand at 37 ° C. for 48 hours. This was designated as Stevioside-lactic acid bacteria treated sample 1. In addition, as a negative control, stevioside was added to PBS (−) containing no lactic acid bacteria so that the final concentration was 40 μg / mL, and the mixture was allowed to stand at 37 ° C. for 48 hours in the same manner. This was designated as Stevioside Sample 1.
<Analysis by HPLC>
The stevioside-lactic acid bacteria treated sample 1 and stevioside sample 1 obtained as described above were centrifuged, and 10 μL of the supernatant was subjected to HPLC under the following conditions. In HPLC (manufactured by Shimadzu Corporation), a reverse phase system using YMC-Pack ODS-A (ID 250 × 4.6 mm, S-5 μm, 12 nm; manufactured by YMC) was used as the separation column, and the mobile phase The analysis was performed under gradient conditions using water and acetonitrile. The column oven was set at 37 ° C. and started with a 10% acetonitrile solution, and the concentration was increased to 100% from 0 to 25 minutes, followed by a washing step with 100% acetonitrile. Detection at 210 nm was performed at a flow rate of 1 mL / min.
The HPLC measurement data of the stevioside-lactic acid bacteria treated sample 1 and stevioside sample 1 are shown in FIGS. 1 and 2, respectively.
＜乳酸菌含有サンプルの調製＞
予め一般乳酸菌接種用培地（ニッスイ社製）に乳酸菌（ラクトバチルス カゼイ ハセガワ菌株）を植え、３７℃で静置培養した後、遠心及びＰＢＳ（−）にて菌体ペレットの洗浄を行い、乳酸菌１×１０１０ｃｆｕを含むＰＢＳ（−）１ｍＬ溶液を１．５ｍＬチューブへ用意した。これを乳酸菌含有サンプルとした。本サンプルを遠心し、上清１０μＬを上述のＨＰＬＣ条件で分析に供した。
乳酸菌含有サンプルのＨＰＬＣ測定データを図３に示す。
<Preparation of lactic acid bacteria-containing sample>
A lactic acid bacterium (Lactobacillus casei Hasegawa strain) is planted in advance in a medium for inoculation of general lactic acid bacteria (Nissui Co., Ltd.), and after standing at 37 ° C., the cell pellets are washed with centrifugation and PBS (−) to obtain lactic acid bacteria 1 A 1 mL solution of PBS (−) containing × 10 10 cfu was prepared in a 1.5 mL tube. This was made into the sample containing lactic acid bacteria. This sample was centrifuged, and 10 μL of the supernatant was subjected to analysis under the above-mentioned HPLC conditions.
The HPLC measurement data of the sample containing lactic acid bacteria is shown in FIG.
＜ルブソシドサンプルの調製＞
市販のルブソシド（和光社製）を予めジメチルスルホキシシド（ＤＭＳＯ）（ナカライテスク社製）に２０ｍｇ／ｍＬの濃度となるように溶解したストック溶液を用意した。乳酸菌を含まないＰＢＳ（−）にルブソシドを最終濃度４０μｇ／ｍＬになるよう加え、３７℃で４８時間静置したものも用意した。これをルブソシドサンプルとした。このサンプル１０μＬを上述のＨＰＬＣ条件で分析に供した。
ルブソシドサンプルのＨＰＬＣ測定データを図４に示す。
図１〜４の結果から、ステビオシドの乳酸菌処理物であるステビオシド−乳酸菌処理サンプル１（図１）では、１４．２min付近のステビオシドのピーク（図２のピーク）が消失し、１５．５min付近のルブソシドのピーク（図４のピーク）が生成したことが確認された。
<Preparation of rubusoside sample>
A stock solution was prepared by previously dissolving commercially available rubusoside (manufactured by Wako) in dimethylsulfoxyside (DMSO) (manufactured by Nacalai Tesque) to a concentration of 20 mg / mL. Rubusoside was added to PBS (-) containing no lactic acid bacteria so that the final concentration was 40 µg / mL, and the mixture was allowed to stand at 37 ° C for 48 hours. This was a rubusoside sample. 10 μL of this sample was subjected to analysis under the above-mentioned HPLC conditions.
The HPLC measurement data of the rubusoside sample is shown in FIG.
From the results of FIGS. 1 to 4, in the stevioside-lactic acid bacterium treated sample 1 (FIG. 1), which is a processed product of stevioside, the stevioside peak (peak in FIG. 2) near 14.2 min disappears, and the vicinity of 15.5 min. It was confirmed that a rubusoside peak (peak in FIG. 4) was generated.
（実施例２）
実施例１において、８０％アセトニトリル溶液をＤＭＳＯに変更した以外は、実施例１と同様の操作を行ない、ステビオシド−乳酸菌処理サンプル２、及びステビオシドサンプル２を調製し、実施例１と同様の方法を用いてＨＰＬＣを測定した。ステビオシド−乳酸菌処理サンプル２、及びステビオシドサンプル２のＨＰＬＣ測定結果を、それぞれ図５及び図６に示す。
これらの図５及び図６の結果からも、乳酸菌を用いたステビオシドの処理により、ステビオシドが消失し、ルブソシドが生成したことが確認された。
(Example 2)
In Example 1, except that the 80% acetonitrile solution was changed to DMSO, the same operation as in Example 1 was performed to prepare Stevioside-lactic acid bacteria treated sample 2 and Stevioside sample 2, and the same method as in Example 1 was performed. Was used to measure HPLC. The HPLC measurement results of the stevioside-lactic acid bacteria treated sample 2 and stevioside sample 2 are shown in FIGS. 5 and 6, respectively.
These results in FIGS. 5 and 6 also confirmed that stevioside disappeared and rubusoside was produced by the treatment of stevioside using lactic acid bacteria.
（実施例３）
実施例１及び２のＨＰＬＣ測定で使用した６種の各サンプル溶液、各６μＬを、ＴＬＣ（薄層クロマトグラフィー）シリカゲルプレート（TLC silica gel 60 F254、メルク社製）へスポットし、移動相をクロロホルム：メタノール：水が15:13:2（v/v/v）の割合となるよう調整し、展開した後、該ＴＬＣシリカゲルプレートを乾燥し、１０％硫酸溶液により噴霧した後、ドライヤーにて発色させた。その写真を図７に示す。
図７において、１はステビオシドサンプル１、２はステビオシドサンプル２、３はルブソシドサンプル、４はステビオシド−乳酸菌処理サンプル１、５はステビオシド−乳酸菌処理サンプル２、６は乳酸菌含有サンプルの展開パターンを示す。
その結果、ステビオシドの乳酸菌処理物である４：ステビオシド−乳酸菌処理サンプル１、及び５：ステビオシド−乳酸菌処理サンプル２は、ルブソシド単体の溶液（３：ルブソシドサンプル）と同じ位置にメインバンドを示し、ステビオシドの乳酸菌処理物が、ルブソシドを主成分として含有することが確認された。
(Example 3)
6 μL of each of the six sample solutions used in the HPLC measurement of Examples 1 and 2 were spotted on a TLC (thin layer chromatography) silica gel plate (TLC silica gel 60 F254, manufactured by Merck), and the mobile phase was chloroform. : Methanol: Water adjusted to a ratio of 15: 13: 2 (v / v / v), developed, dried TLC silica gel plate, sprayed with 10% sulfuric acid solution, developed color with dryer I let you. The photograph is shown in FIG.
In FIG. 7, 1 is a stevioside sample 1, 2 is a stevioside sample 2, 3 is a rubusoside sample, 4 is a stevioside-lactic acid bacteria treated sample 1, 5 is a stevioside-lactic acid bacteria treated sample 2, and 6 is a development pattern of a sample containing lactic acid bacteria. Show.
As a result, stevioside-treated lactic acid bacteria treated sample 4: stevioside-lactic acid bacteria treated sample 1 and 5: stevioside-lactic acid bacteria treated sample 2 showed a main band at the same position as the solution of rubusoside alone (3: rubusoside sample). It was confirmed that the processed product of stevioside lactic acid bacteria contains rubusoside as a main component.
（実施例４）
＜ステビア抽出物の調製＞
ステビア葉の乾燥粉末を１０ｇ測りとり、そこへ蒸留水を１００ｍＬ加え、撹拌しながら８０℃で３時間抽出を行った。その後、Ｎｏ．２のろ紙（アドバンテック社製）で濾過し、得られた溶液を凍結乾燥へと供した。
＜ステビア抽出物―乳酸菌処理サンプルの調製＞
予め一般乳酸菌接種用培地（ニッスイ社製）に乳酸菌（ラクトバチルス カゼイ ハセガワ菌株）を植え、３７℃で静置培養した後、遠心及びＰＢＳ（−）にて菌体ペレットの洗浄を行い、乳酸菌１×１０１０ｃｆｕを含むＰＢＳ（−）１ｍＬ溶液を１．５ｍＬチューブへ用意した。一方で上記のステビア抽出物を予め滅菌蒸留水（Ｈ２Ｏ）に４０ｍｇ／ｍＬの濃度となるように溶解したストック溶液を用意した。該ストック溶液を0.22μｍ滅菌フィルターを通して滅菌し、乳酸菌菌体が含まれているＰＢＳ（−）溶液へステビア抽出物の最終濃度が８ｍｇ／ｍＬになるように加え、３７℃で９６時間静置した。これをステビア抽出物―乳酸菌処理サンプルとした。
＜ＨＰＬＣによる分析＞
上記の通り得られたステビア抽出物―乳酸菌処理サンプルを遠心し、上清１０μＬを以下の条件下でＨＰＬＣへ供した。ＨＰＬＣ（島津製作所社製）では分離カラムにＹＭＣ−Ｐａｃｋ ＯＤＳ−Ａ （Ｉ．Ｄ． ２５０×４．６ｍｍ、Ｓ−５μｍ、１２ｎｍ；ＹＭＣ社製）を用いた逆相系を使用し、移動相として水及びアセトニトリルを用いたグラジエント条件下で分析を行った。カラムオーブンを３７℃に設定したまま１０％アセトニトリル溶液から開始し、０分から２５分までに１００％まで濃度を上げ、その後１００％アセトニトリルによる洗浄工程を経る条件を用いた。流速１ｍＬ／分で２１０ｎｍでの検出を行った。
ステビア抽出物―乳酸菌処理サンプルのＨＰＬＣ測定データを図８に示す。
Example 4
<Preparation of stevia extract>
10 g of dry powder of stevia leaf was measured, 100 mL of distilled water was added thereto, and extraction was performed at 80 ° C. for 3 hours with stirring. Then, no. The mixture was filtered through No. 2 filter paper (manufactured by Advantech), and the resulting solution was subjected to lyophilization.
<Stevia extract-Preparation of lactic acid bacteria treated sample>
A lactic acid bacterium (Lactobacillus casei Hasegawa strain) is planted in advance in a medium for inoculation of general lactic acid bacteria (Nissui Co., Ltd.), and after standing at 37 ° C., the cell pellets are washed with centrifugation and PBS (−) to obtain lactic acid bacteria 1 A 1 mL solution of PBS (−) containing × 10 10 cfu was prepared in a 1.5 mL tube. On the other hand, a stock solution in which the above stevia extract was previously dissolved in sterile distilled water (H 2 O) to a concentration of 40 mg / mL was prepared. The stock solution was sterilized through a 0.22 μm sterilizing filter, added to a PBS (−) solution containing lactic acid bacteria so that the final concentration of stevia extract was 8 mg / mL, and allowed to stand at 37 ° C. for 96 hours. . This was used as a stevia extract-treated with lactic acid bacteria.
<Analysis by HPLC>
The stevia extract-lactic acid bacteria-treated sample obtained as described above was centrifuged, and 10 μL of the supernatant was subjected to HPLC under the following conditions. In HPLC (manufactured by Shimadzu Corporation), a reverse phase system using YMC-Pack ODS-A (ID 250 × 4.6 mm, S-5 μm, 12 nm; manufactured by YMC) was used as the separation column, and the mobile phase The analysis was performed under gradient conditions using water and acetonitrile. The column oven was set at 37 ° C. and started with a 10% acetonitrile solution, and the concentration was increased to 100% from 0 to 25 minutes, followed by a washing step with 100% acetonitrile. Detection at 210 nm was performed at a flow rate of 1 mL / min.
FIG. 8 shows the HPLC measurement data of the stevia extract-treated lactic acid bacteria sample.
（比較例１）
＜ステビア抽出物サンプルの調製＞
ネガティブコントロールとして、乳酸菌を含まないＰＢＳ（−）に前記ステビア抽出物を最終濃度８ｍｇ／ｍＬになるよう加え、同様に３７℃で９６時間静置したものも用意した。これをステビア抽出物サンプルとした。該サンプルを実施例４と同様の方法を用いてＨＰＬＣ測定を行った。
ステビア抽出物サンプルのＨＰＬＣ測定データを図８に示す。
図８の結果から、比較例１のステビア抽出物サンプルに含有される１４．２min付近のステビオシドのピークが、実施例４のステビア抽出物―乳酸菌処理サンプルでは減少しており、実施例４のピークでは１５．５min付近のルブソシドのピークが見られた。従って、ステビア抽出物に含有されるステビオシドを、乳酸菌を用いて処理することにより、ルブソシドを製造することができることが示された。
(Comparative Example 1)
<Preparation of stevia extract sample>
As a negative control, the stevia extract was added to PBS (−) containing no lactic acid bacteria so as to have a final concentration of 8 mg / mL, and the mixture was also allowed to stand at 37 ° C. for 96 hours. This was used as a stevia extract sample. The sample was subjected to HPLC measurement using the same method as in Example 4.
The HPLC measurement data of the stevia extract sample is shown in FIG.
From the results of FIG. 8, the peak of stevioside in the vicinity of 14.2 min contained in the stevia extract sample of comparative example 1 is decreased in the stevia extract-treated sample of lactic acid bacteria of example 4, and the peak of example 4 Then, a rubusoside peak around 15.5 min was observed. Therefore, it was shown that rubusoside can be produced by treating stevioside contained in stevia extract with lactic acid bacteria.
本発明のルブソシドの製造方法によれば、食経験上十分に安全な原料を使用して、食しても十分に安全なルブソシドを、安価で簡便に製造することができ、大量生産することができるため、ルブソシドを甘味料や難溶解性薬剤の天然可溶化剤として実用化することができる。 According to the method for producing rubusoside of the present invention, it is possible to easily and inexpensively produce rubusoside, which is sufficiently safe even if eaten, using raw materials that are sufficiently safe in eating experience, and can be mass-produced. Therefore, rubusoside can be put into practical use as a natural solubilizer for sweeteners and poorly soluble drugs.
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