JPS59120073A - Sweetener and its preparation - Google Patents
Sweetener and its preparationInfo
- Publication number
- JPS59120073A JPS59120073A JP57229417A JP22941782A JPS59120073A JP S59120073 A JPS59120073 A JP S59120073A JP 57229417 A JP57229417 A JP 57229417A JP 22941782 A JP22941782 A JP 22941782A JP S59120073 A JPS59120073 A JP S59120073A
- Authority
- JP
- Japan
- Prior art keywords
- stevioside
- rebaudioside
- reaction
- enzyme
- sweetener
- 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.)
- Pending
Links
Landscapes
- Seasonings (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔発明の概要と背最〕
本発明は新しい修飾天然は法科、さらに詳しくは、ステ
ビオサイドやしlくウデイオサイドーA力)ら生化学的
手法によって生成さオtたβ−り゛1ノコシルステビオ
サイド類やβ−り゛1ノコシルレノくウデイオサイドー
A碩を含有する甘味料ノシひその製j告法に関する。[Detailed Description of the Invention] [Summary and Background of the Invention] The present invention relates to a new modified natural product, more specifically, a novel modified natural β- The present invention relates to a method for producing a sweetener containing ri-1-nocosyl steviosides and β-1-nocosyl steviosides.
ステビア(5tevia Rebaudiana Be
rtoni ) iま、南米パラグアイ原産のキク科多
年生、値物であり、その葉中には、ステビオサイドを主
体とするジテルペン系配糖体甘味物質1が含まれてしす
るので、これを抽出・精製し、ステビア抽出物又Gマス
テビア抽出精製物として甘味料に供するものである。Stevia (5tevia Rebaudiana Be
It is a valuable perennial of the Asteraceae family that is native to Paraguay in South America, and its leaves contain diterpene-based glycoside sweet substances 1, mainly stevioside, which are extracted and purified. It is used as a sweetener as a stevia extract or a purified G-mastevia extract.
*ステビア葉中の配糖体甘味物質として(よ、ステビオ
サイドの仲、レノくウデイオサイド−A、c、0.h、
及びズルコサイドーAの各成分りく知られている。* As a glycoside sweet substance in Stevia leaves (Yo, a member of Stevioside, Lenoku Udeioside-A, C, 0.h,
The components of zulcoside A and zulcoside A are well known.
一般に、ステビオサイドはステヒ゛ア葉中の内己塘体は
味成分のうち含量的に般も多く、ステビオサイド・ステ
ビア抽出精製物中の甘味成分の主体をなしている成分で
ある。本島Cま、甘味質力)比咬B勺砂糖に近く、甘味
倍数も砂糖の約300倍と高し′1力S、反面、苦味や
渋味をも有するため、これらの不快味が日中に残る欠点
がある。In general, stevioside is present in the internal body of Stevia leaves in terms of its content in terms of taste components, and is the main component of the sweetness component in the purified stevioside/stevia extract. Main island C, sweetness quality) Hibuki B is similar to sugar, and has a sweetness factor about 300 times higher than sugar, but on the other hand, it also has a bitter and astringent taste, so these unpleasant tastes are absorbed during the day. There are some remaining drawbacks.
レバウディオサイド−Aは、含量的にステビオサイドに
次いて多い成分てあり、甘味度はステビオサイドより高
く、しかも、苦味・渋味を待たず、これらの不快味を日
中に残さない良質のけ味成分である。Rebaudioside-A is the second-largest component after stevioside in terms of content, has a higher sweetness than stevioside, and is a high-quality substitute that does not have a bitter or astringent taste and does not leave these unpleasant tastes during the day. It is a flavor component.
レバウディオサイド−CとズルコサイドーAとは、とも
にラムノースを有する成分であるが、甘味強度は、それ
ぞれレバウディオサイド−A及びステビオサイドの約1
/10程度に過ぎず、含量的にも少量であるから甘味料
の対象とはなり雉い。、レバウディオサイド−〇、Eは
、ともに虻味強度・甘味質ともステビオサイドより優れ
ていると評価されているが、微量成分であるため甘味料
の対象となり難い。以下、・参考までにこれまでに確認
されている6種のステビアオ味配糖体成分の化学構造を
示す。Rebaudioside-C and dulcoside-A are both components that contain rhamnose, but the sweetness intensity is about 1 that of rebaudioside-A and stevioside, respectively.
It is only about /10, and the content is so small that it cannot be used as a sweetener. , Rebaudioside-〇, and E are both evaluated to be superior to stevioside in terms of meaty taste intensity and sweetness quality, but since they are trace components, they are difficult to be used as sweeteners. Below, for reference, the chemical structures of six types of Stevia flavor glycoside components that have been confirmed so far are shown.
−、、、J 4 “。、
Wロ ′ −1゛
ステビア葉中に含有されていることが認められディオサ
イド−Aは、甘味の強さ、質ともにステビオサイドより
優れており、含量的にもステビオサイドに次いて多いた
め、ステビア抽出物・ステビア抽出精製物の甘味に重要
な役割を持つ成分である。(7たかつて、ステビア配糖
体甘味成分のうち、甘味料として最も注目されている成
分である。-,,,J 4 "., Wro'-1" Dioside-A, which is found to be contained in stevia leaves, is superior to stevioside in both sweetness strength and quality, and also in terms of content. It is the second most abundant component after stevioside, and therefore plays an important role in the sweetness of stevia extracts and purified stevia extracts. It is.
このレバウディオサイド−Aをステビア葉中から抽出し
、精製・単11jlu してけ法科に供することも可能
であるか、
■ 精製コストが高くなる。Is it possible to extract this Rebaudioside-A from Stevia leaves, purify it, and then use it for legal purposes? (1) Purification costs will be high.
■ 含量的に最も多いステビオサイド画分が副産物とし
て多隈に得られ、しかも、苦味・渋味が分・離削のもの
より強くなるため、甘味料として利用しにくくなる。■ The stevioside fraction, which has the highest content, is obtained in large quantities as a by-product, and moreover, it has a stronger bitterness and astringency than the stevioside fraction, making it difficult to use as a sweetener.
等のデメリ/トか生じるので、これを工業的に実施する
意味が薄れる。Since such disadvantages/disadvantages arise, it becomes less meaningful to implement this method industrially.
また、レバウディオサイド−Aをステビオサイドから化
学合成することも研究され、実鹸的には可能であること
が認められている。しかし−,1テ、、、、y合成では
、
■ 反応工程か多く、しかも、複雑である。Further, chemical synthesis of rebaudioside-A from stevioside has been studied, and it has been recognized that it is actually possible. However, -,1te,...,y synthesis requires a large number of reaction steps and is complicated.
■ 得られた製品は、合成甘味料と児なされ、天然甘味
料のような利用かでき難い。■ The resulting product is considered a synthetic sweetener and cannot be used like a natural sweetener.
等の欠点かある。There are some drawbacks such as.
そこで、本発明者は、ステビオサイドを原料として、レ
バウディオサイド−Aを酵素を用いて合成すへく研究を
開始したが、その結果、ステビオサイドやレバウディオ
サイド−Aに、β−クルコシル糖化合物を基質としてβ
−グルコシル転移酵素を作用させて、β−グリコジルス
テビオサイドやθ−グリコジルレバウディオサイド−A
に変化させうることを見出し、これらのβ−グリコジル
誘導体は、苦味・渋味や不快な渋味かなく非常に良質の
け味であることをも見出した。Therefore, the present inventor started research on synthesizing rebaudioside-A using enzymes using stevioside as a raw material. β using a compound as a substrate
-By acting glucosyltransferase, β-glycodyl stevioside and θ-glycodyl rebaudioside-A
They also found that these β-glycodyl derivatives have a very good quality of flavor, without bitterness, astringency, or unpleasant astringency.
すなわち、本発明は、ステビア甘味成分を酵素化学反応
の利用によって、β−グリコジル誘導体に変換し、ステ
ビア甘味料の欠点である苦味・渋味をなくし、不快な渋
味を感じさせない良質甘味に改善することを木質とする
ものである。That is, the present invention converts stevia sweetness components into β-glycodyl derivatives using enzymatic chemical reactions, eliminates the bitterness and astringency that are the drawbacks of stevia sweeteners, and improves the quality of sweetness without unpleasant astringency. It is made of wood.
木発明者は、本発明にかかわる酵素反応によって、ステ
ビオサイドが、β−グリコジルステビオサイド類に変換
していることを確認し、レバウディオサイド−Aにも本
発明による反応が進行することを確認した。The inventors have confirmed that stevioside is converted into β-glycodyl steviosides by the enzymatic reaction of the present invention, and that the reaction of the present invention also progresses to rebaudioside-A. did.
本発明における酵素反応は、高度に精製されたステビオ
サイドやレバウディオサイドー八に限るものではなく、
両者の混合物であっても、さらにはステビア抽出物・ス
テビア抽出精製物であってもよい。要は、原料物質が゛
ステビオサイドやレバウディオサイド−へのような成分
を含有する限り、これらの成分はβ−グリコジル誘導体
に変換され、本発明による甘味料が製造できる。The enzymatic reaction in the present invention is not limited to highly purified stevioside and rebaudioside,
It may be a mixture of both, or it may be a stevia extract or a purified stevia extract. In short, as long as the raw material contains components such as stevioside and rebaudioside, these components can be converted into β-glycodyl derivatives to produce the sweetener according to the present invention.
本発明に用いる基質は、β〜グリコジル糖化合物である
が、例えばフラボバクテリウム・ドルミテーターの生産
するβ−グルコシル転移酵素の場合であれば、ラミナリ
ン・パキマン・酵母グルカン・リケナン・カードラン等
が用いられる。The substrate used in the present invention is a β-glycosyl sugar compound. For example, in the case of β-glucosyltransferase produced by Flavobacterium dormitator, laminarin, pachyman, yeast glucan, lichenan, curdlan, etc. are used. It will be done.
また、本発明に用いられるβ−グルコシル転移酵素は、
必ずしも精製されている必要はなく、精製途中段階の酵
素であっても、さらには培養渥液であっても充分に目的
を達することができる。Furthermore, the β-glucosyltransferase used in the present invention is
The enzyme does not necessarily need to be purified, and even the enzyme in the middle of purification, or even the culture sap, can sufficiently achieve the purpose.
本発明の反応は、ステビア配糖体甘味成分とβ−グルコ
シル糖化合物とを含有する水・容液に、β−グルコシル
転移酵素を作用させることによって行われるが、より好
ましくは、酵素の最適反応条件、又はそれに近い条件が
選ばれる。すなわち、温度30〜60℃、PH4,5〜
7,5 にて反応時間2〜100時間とするのがよい
。The reaction of the present invention is carried out by allowing β-glucosyltransferase to act on water/solution containing a stevia glycoside sweet component and a β-glucosyl sugar compound. The condition or a condition close to it is selected. That is, temperature 30~60℃, PH4.5~
7,5 and the reaction time is preferably 2 to 100 hours.
また、反応に用いられるステビア配糖体甘味成分とβ−
グルコシル糖化合物との割合は、特に制限されるもので
はないが、好ましくはステビア配塘体片味成分の重量に
対するβ−グルコシル糖化合物の重量比率を0.25〜
100 の範囲とするのがよい。さらに、反応液中の
ステビオサイドとβ−グルコシル糖化合物との固形分濃
度についても特に制限はないが、溶解性の点から1〜8
0%とするのがよい。In addition, the sweet component of stevia glycoside used in the reaction and β-
The ratio of the β-glucosyl sugar compound to the weight of the component of the Stevia package is preferably 0.25 to 0.25, although the ratio of the β-glucosyl sugar compound to the weight of the component of the Stevia package is not particularly limited.
A range of 100 is recommended. Furthermore, there is no particular restriction on the solid concentration of stevioside and β-glucosyl sugar compound in the reaction solution, but from the viewpoint of solubility, it is
It is better to set it to 0%.
本発明による反応の実施前後で甘味強度はほとんど同じ
である。しかし、甘味質は反応の前後でいちじるしく異
なり、反応後では反応前に見られた苦味・渋味か消失し
、しかも、不快な残株を呈しない、されやかでまろやか
な良質甘味となる。The sweetness intensity is almost the same before and after carrying out the reaction according to the invention. However, the quality of the sweetness differs significantly before and after the reaction, and after the reaction, the bitterness and astringency seen before the reaction disappear, and moreover, the sweetness becomes smooth, mellow, and of high quality without any unpleasant residue.
このように、本発明の反応によって生成されたβ−グリ
コジル誘導体・を含有する反応溶液は、そのままでもけ
法科として使用できるが、必要に応じて、未反応の基質
をr別し、さらに、イオン交換樹脂を用いて脱塩し、濃
縮して液状製品とすることもできる。また、これを乾燥
・粉末化して粉末製品とすることもできるし、造粒し、
顆粒製品とすることもできる。As described above, the reaction solution containing the β-glycodyl derivative produced by the reaction of the present invention can be used as it is as a chemical solution, but if necessary, unreacted substrates are separated and ions are added. It can also be desalted using an exchange resin and concentrated into a liquid product. In addition, it can be dried and powdered to make a powder product, or it can be granulated,
It can also be a granule product.
このようにして得られる本発明の甘味料は、ステビオサ
イドに特有な呈味の欠点が改善され、されやかでまろや
かな良質甘味となるため、食品及び医薬品の甘味料とし
て特に好ましいものである。The sweetener of the present invention obtained in this manner is particularly preferable as a sweetener for foods and medicines because it improves the taste defects peculiar to stevioside and provides a smooth, mellow, high-quality sweet taste.
以下、実験によって本発明を説明するが、本発明の技術
的範囲は、これらによって何ら限定されるものではない
。The present invention will be explained below through experiments, but the technical scope of the present invention is not limited by these in any way.
、実験1 甘味料の製造
1−1 酵素の調製
フラボバクテリウム ドルミテーター バルグルカノリ
ティヵ(Flavobacterium dortni
tatorvar gl ucariol yt 1c
ae ) を酵母4%、 KH21’040.3%、
FeSO4°71(200,07%かラナル殺菌シタ
培地CPH8,7) 3.000rd (5000−容
シ+ −7y −メンタ−)に17白金耳植菌し、30
”C,72時間通気攪拌培養した。培養後、培養液を1
3. OOO’xG S分間遠心分離し、透明な培養r
液を得た。涙液は、減圧下に30℃で約1/1oに濃縮
し、硫安20〜80%飽和区分で塩析した。塩析した酵
素は、10,000xG 5分間遠心により集め、0
..01 iν1 リン酸カリウム’(K −Phos
phate )緩衝液、 pH5,0ニ溶解し、開成に
て、5℃1夜透析した。, Experiment 1 Production of sweetener 1-1 Preparation of enzyme Flavobacterium dortni.
tatorvar gl ucariol yt 1c
ae) with yeast 4%, KH21'040.3%,
FeSO4°71 (200.07% or Ranal sterilized Shita medium CPH8.7) 17 platinum loops were inoculated into 3.000rd (5000-volume Shi+-7y-mentor), 30
"C. Cultured with aeration for 72 hours. After culture, the culture solution was
3. Centrifuge for OOO'xG S to clear culture r
I got the liquid. The lachrymal fluid was concentrated to about 1/1 degree under reduced pressure at 30° C. and salted out with ammonium sulfate at 20-80% saturation. The salted out enzyme was collected by centrifugation at 10,000xG for 5 minutes and
.. .. 01 iν1 Potassium phosphate' (K-Phos
phate) buffer, pH 5.0, and dialyzed overnight at 5°C.
透析液(酵素1)をQ、 QI M IJン酸カリウ
ム緩衝液、p+−15,0にて平衡にした。SE−セフ
ァデックス C−50カラムに通じ、吸着した酵素をo
、olra リン酸カリウム緩衝液、Pt(5,Qに
て溶出させた。活性の強い区分をアミコン(Amjco
n)PM −10メンブランフィルタ−を用いて濃縮し
、濃縮液はバイオゲル(13io Gel ) −P
150カラムにでゲルρ過した。ゲルr過1−た酵素を
電気体動にて純度を検定したところ均一な標品が得られ
なかったので、さらにSP−セファデックス(SeFI
hadex ) C−50カラムによ−って再クロマド
グと
ラフイーを行って濃縮、ディスク電気泳動的に均一な酵
素標品を得た。(酵素2)
なお、酵素2の最終液射は、九−で、そのタンパク・C
a度は、牛血清アルブミンを標準品として280個の吸
光度を測定した結果、1.1 Jnf/rriてあった
。また、酵素2の活性は、β−1,3−グルカナーゼの
活性として、132 unit /mlであった。ここ
でいう活性1 unitとは、Q、 l +J コハク
酸ナトリウム緩衝液、)’)I 5.8 、 に溶解
したラミナリン溶液(5my/m!、 )の0.5m!
に、適当に希釈した酵素液0.5 dを加えて全量を1
mlとし、40℃、10分間反応させた後、ソモギー(
Somogyi )試薬1−を加えて反応を停止させ、
生成した還元糖をソモギー・ネ/l/ 77 (Som
ogyi −Ne1son )法で定量したとき、10
分間に1μmoleの還元糖(グルコースとして)を生
成させる酵素量をいう。The dialysate (enzyme 1) was equilibrated with Q, QIM IJ potassium phosphate buffer, p+-15,0. Passed through a SE-Sephadex C-50 column to remove the adsorbed enzyme.
, olra potassium phosphate buffer, Pt (5,Q).
n) Concentrate using a PM-10 membrane filter, and the concentrated solution is biogel (13io Gel)-P.
The gel was filtered through a 150 column. When the purity of the gel-filtered enzyme was tested using electrodynamics, a homogeneous sample could not be obtained, so SP-Sephadex (SeFI
The enzyme was concentrated by rechromatography and roughy using a Hadex C-50 column, and an enzyme preparation homogeneous in disk electrophoresis was obtained. (Enzyme 2) The final injection of Enzyme 2 is 9-, and its protein and C
The a degree was 1.1 Jnf/rri as a result of measuring the absorbance of 280 samples using bovine serum albumin as a standard. Furthermore, the activity of enzyme 2 was 132 units/ml as the activity of β-1,3-glucanase. Here, 1 unit of activity means 0.5 m! of a laminarin solution (5 my/m!, ) dissolved in Q, l + J sodium succinate buffer, )') I 5.8, !
Add 0.5 d of appropriately diluted enzyme solution to bring the total volume to 1
After reacting at 40°C for 10 minutes, Somogyi (
Somogyi) reagent 1- is added to stop the reaction,
The resulting reducing sugar is converted to Somogyi Ne/l/77 (Som
ogyi-Ne1son) method, 10
It refers to the amount of enzyme that produces 1 μmole of reducing sugar (as glucose) per minute.
22.000 (BioGel P −150ケルl
過)25.000 (SO8電気永動 )28
.000 (超遠心ただL S 20W= 3.46
)偏比容値 0.75 cc/りとした。22.000 (BioGel P-150 Kell
25.000 (SO8 Denki Eido) 28
.. 000 (Ultracentrifuge only L S 20W = 3.46
) The partial specific volume value was set to 0.75 cc/liter.
超遠心法で求めた分子量28. OOOを用いてアミノ
酸の個数を算出した結果、234g1のアミノ酸から成
り立っていることがわかった(表19゜また、この組成
から偏比容値は0.711 cc、/g′ が求めら
れ、この値では前述の超遠心法による分子量は約24,
000となる。Molecular weight determined by ultracentrifugation 28. As a result of calculating the number of amino acids using OOO, it was found that it was made up of 234g1 of amino acids (Table 19゜). Also, from this composition, the partial specific volume value was determined to be 0.711 cc, /g', and this The molecular weight according to the ultracentrifugation method described above is approximately 24,
It becomes 000.
(以下余白)
表 1
〔基質特異性〕
種々な基質を用いて酵素2の特異性を調べた結果を表2
に示す。(Left below) Table 1 [Substrate specificity] Table 2 shows the results of investigating the specificity of enzyme 2 using various substrates.
Shown below.
表2から、本酵素はラミナリン、パキマン、イーストグ
ルカン、リケナンなどのβ−1,3結合を有する基質に
のみ作用し、還元糖を生成することかわかる。Table 2 shows that this enzyme acts only on substrates having β-1,3 bonds, such as laminarin, pachyman, yeast glucan, and lichenan, and produces reducing sugars.
この結果から、本酵素ばβ−1,3−ダルカナーゼであ
ると結論される。From this result, it is concluded that the enzyme is β-1,3-dalcanase.
酵素2の最適反応条件を下記に示す。 The optimal reaction conditions for Enzyme 2 are shown below.
最適温度 45〜50℃
最適ピHPH5,5〜65
温度安定性 50℃まで安定しH安
定性 μH6,0〜 9.0〔糖転移反応〕
最終濃度が100’mMと5mMになるようにP−二ト
I:’7エ=/レーβ−D−ゲルコンド(P−Ni t
ro’pheny I−β−D −glucosid
e )およびラミナリンをそれぞれ0.OIM IJン
酸カリウム緩衝液、PH60に溶解し、酵素2を1μL
(11μ2)加え、50℃、20分間反応させた。反応
後、反応液50μtを東洋F紙高50にスポットし、n
−ブタノール:エタノール:水−5,:2:2の展開液
にて下降法で展開した。E紙を風乾し、紫外線ランプに
て反応生成物のスポット位置を確認した。Optimum temperature 45-50℃ Optimum pH 5.5-65 Temperature stability Stable up to 50℃, H stability μH 6.0-9.0 [Glucose transfer reaction] P- Nito I:'7E=/Le β-D-Gelcondo (P-Nit
ro'pheny I-β-D-glucosid
e) and laminarin at 0.0. Dissolve 1 μL of enzyme 2 in OIM IJ potassium acid buffer, PH60.
(11μ2) and reacted at 50°C for 20 minutes. After the reaction, 50 μt of the reaction solution was spotted on Toyo F paper height 50, and
-Butanol:Ethanol:Water-5: It was developed in a descending method using a developing solution of 2:2. Paper E was air-dried, and the spot position of the reaction product was confirmed using an ultraviolet lamp.
第1図に酵素を加えずに上記と同様処理した液と酵素を
加えて反応された液のクロマトクラムを示す。FIG. 1 shows chromatograms of a solution treated in the same manner as above without adding an enzyme and a solution reacted with the addition of an enzyme.
図面から、酵素を加えて反応させた液には、未反応のP
−二トロフェニルーβ−D−クルコシドの他に糖の転移
によって生成した多数の反応生成物のスポットが見られ
る。From the drawing, it can be seen that unreacted P is present in the solution in which the enzyme is added and reacted.
In addition to -nitrophenyl-β-D-curcoside, numerous spots of reaction products produced by sugar transfer can be seen.
この結果、本酵素が糖転移作用を有することがわかる。The results show that this enzyme has glycosyltransfer activity.
1だ、この糖転移は、酵素の基質特異性からβ−1,3
−結合で糖の転移を生ずるものと結論される。1, this sugar transfer occurs due to the substrate specificity of the enzyme.
It is concluded that - binding causes sugar transfer.
1−3 甘味料の製造
薄層クロマトクラム及び高速液体クロマトグラムにてス
テビオサイド以外のステビア系配糖体甘味成分のスポッ
トやピークが検出されなくなるまでに高度に精製された
ステビオサイド12を0.01Mリン酸カリウム緩衝液
、PH6,0、50m1に加熱溶解し、さらに、ラミナ
リン12を加えて溶解した。1-3 Production of sweetener Highly purified stevioside 12 is added to 0.01 M phosphorus until spots and peaks of sweet components of stevia-based glycosides other than stevioside are no longer detected in thin-layer chromatograms and high-performance liquid chromatograms. The mixture was heated and dissolved in 50 ml of acid potassium buffer, pH 6.0, and then laminarin 12 was added and dissolved.
この液に、酵素2を0.5 rnl加えて50℃にて1
夜反応させた。反応液を沸騰水中に10分間保って酵素
を失活させ、液状甘味料を得た。(試料1)第2図に試
料1の薄層クロマトグラムを示す。Add 0.5 rnl of enzyme 2 to this solution and heat at 50℃ for 1 hour.
I reacted at night. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme and obtain a liquid sweetener. (Sample 1) The thin layer chromatogram of Sample 1 is shown in FIG.
なお、N層りロマトグラフィーは、HPTLC用シリカ
デシリカゲル60薄
溶媒700ホルム:メタノ−)v:水=30:20:4
にて展開し、風乾した後、20%硫酸を噴霧.加熱して
発色させた。For N layer chromatography, HPTLC silica gel 60 thin solvent 700 form:methanol)v:water=30:20:4
After developing and air drying, 20% sulfuric acid was sprayed. It was heated to develop color.
図面から、本発明の酵素反応によって、ステビオサイド
から新規反応生成物が得られていることは明白である。From the figures, it is clear that novel reaction products are obtained from stevioside by the enzymatic reaction of the present invention.
また、わずかではあるが本反応によってレバウディオサ
イド−Aが生成されていることがわかる。これは、本発
明に使用する酵素が糖転移作用を有するβ−】、3−ク
ルヵナーゼであることを考えればもっともなことである
と考えられる。It is also seen that rebaudioside-A is produced by this reaction, albeit in a small amount. This is considered to be reasonable considering that the enzyme used in the present invention is β-],3-curcanase which has glycosyltransfer activity.
実験2 反応生成物の精製
実験1−3で得た試料1を合成吸着樹脂HP−5020
0 fnlをつめだカラムにSVIで通じ、未反応のス
テビオサイド及び反応生成物を吸着せしめた後、カラム
を水i充分洗浄して糖質や塩類を除去した。Experiment 2 Purification of reaction product Sample 1 obtained in Experiment 1-3 was purified using synthetic adsorption resin HP-5020.
After passing 0 fnl through a column packed with SVI to adsorb unreacted stevioside and reaction products, the column was thoroughly washed with water to remove carbohydrates and salts.
次に、アセトン:水=1=1の混合溶媒500−を用い
て吸着物を溶出させ、液出液を濃縮.乾固して約1.3
2の粉末状甘味料を得だ。(試料2)試料2の12をシ
リカゲルカラムクロマトグラフィー(ワコーゲルc −
2(10 、φ2.j×95 cm 、展開’f8
tiX ハクロロホルム:メタノール: 水= 80
:20:4)にて分画し、試料3を200m1F得た。Next, the adsorbate was eluted using a mixed solvent of acetone:water=1=1, and the eluate was concentrated. Dry to about 1.3
You will get 2 powdered sweeteners. (Sample 2) 12 of sample 2 was subjected to silica gel column chromatography (Wako gel c-
2 (10, φ2.j x 95 cm, unfolded 'f8
tiX hachloroform: methanol: water = 80
:20:4) to obtain 200 ml of sample 3.
試料3の薄層クロマトグラムを第3図に示す。The thin layer chromatogram of sample 3 is shown in FIG.
図面から、試料8は高度に精製されていることがわかる
。また、レバウディオサイド−Aとは異なる物質である
こともわかる。From the drawing, it can be seen that sample 8 is highly purified. It can also be seen that it is a different substance from rebaudioside-A.
一方、第2図から、試料3以外の反応生成物が存在して
いることは明白であるが、量的に少ないためか分画でき
なかった。On the other hand, from FIG. 2, it is clear that reaction products other than sample 3 are present, but they could not be fractionated, probably because they were small in quantity.
実験3 試料3の構造決定
試料3は、レバウディオサイド−Aとは異なる物質であ
り、薄層クロマトグラムからステビオサイドにグルコー
ス1残基が結合している配糖体であると考えられる。Experiment 3 Structure Determination of Sample 3 Sample 3 is a substance different from rebaudioside-A, and is considered to be a glycoside in which one glucose residue is bound to stevioside from the thin layer chromatogram.
一方、本反応に使用した酵素20基質特異性は、β−1
,8−結合に特異的に作用することがら、ステビオサイ
ドへの糖転移サイドは、下図のようにしかるに、試料8
の薄層クロマトグラムのスポット位置から、レバウディ
オサイド−Aとは異なることがわかっているので■では
ないことは明白である。On the other hand, the substrate specificity of the enzyme 20 used in this reaction was β-1
, 8 - Since the sugar transfer side to stevioside acts specifically on the bond, as shown in the figure below, sample 8
Since it is known from the spot position of the thin layer chromatogram that it is different from Rebaudioside-A, it is clear that it is not ■.
そこで、■であるか■であるがを確かめるために、アル
カリ加水分解してc16−に結合しているグルコーヌ残
基金化学的に切って薄層り巨マドグラフィーを行ってみ
た。もし、■であればヌテビオールビオザイド(Ste
violbioside)の位置にスポットを生じるは
ずであり、■であれば別の位置にスポットが生じるはず
である。その結果を第4図に示す。Therefore, in order to confirm whether the condition is ``■'' or ``■'', the residual glucone bonded to c16- after alkali hydrolysis was chemically cut and thin-layer macromathography was performed. If ■, Nuteviol biozide (Ste
A spot should be generated at the position (violbioside), and a spot should be generated at another position if it is ■. The results are shown in FIG.
図面から、試料3のアルカリ加水分解物のスポット位置
は、レバウディオサイド−Bの位置に非常に近いが、わ
ずかに下にずれていることがわかる。すなわち、レバウ
ディオサイド−Bとは異なる新物質であると考えられる
。From the drawing, it can be seen that the spot position of the alkaline hydrolyzate of Sample 3 is very close to the position of Rebaudioside-B, but slightly shifted downward. In other words, it is considered to be a new substance different from rebaudioside-B.
この結果から、試料3の化学構造は、ステビオサイドの
■■■の転移サイトのうちの■の位置にクルコーヌ1残
基がβ−1,3−結合しているものと考えられる。From this result, it is considered that the chemical structure of Sample 3 is that Curcone 1 residue is β-1,3-bonded to the position of ■ of the ■■■ transfer site of stevioside.
第5図に、試料3の炭素13−核磁気共鳴スペクトルを
示す。試料は、ピリジンi中、100■/mlの濃度で
室温にて、パルスFT−幅仏ヌベク)/しを測定した。FIG. 5 shows the carbon-13 nuclear magnetic resonance spectrum of Sample 3. Samples were measured for pulse FT-width in pyridine at a concentration of 100 ml/ml at room temperature.
化学シフトは、ピリジンdiの3組の1−リプレットシ
グナルの中央のシグナルを各々123.6185.21
49.8 ppmとして8 ppmで示した。その結果
、ステビオサイドのC−20の化学シフトは、15、9
ppm (文献値15.4’ ppm )、C−19
では17’f、8ppm (文献値177、0 ppm
)が得られたので化学シフトの求め方は正しいものであ
ると考えられた。The chemical shift is 123.6185.21 for each of the central signals of the three 1-replet signals of pyridine di.
It was expressed as 8 ppm as 49.8 ppm. As a result, the chemical shift of C-20 of stevioside is 15,9
ppm (literature value 15.4' ppm), C-19
So 17'f, 8 ppm (literature value 177, 0 ppm
) was obtained, so the method for determining the chemical shift was considered to be correct.
図面に示した炭素−13核磁気共鳴ヌベクl−/しから
、98.1 ppmにC−18−OHに結合したβ−D
−グルコーヌのアノマー゛炭素、 96.2 ppmK
(、−19ニス1、チル型クルコースのアノマー炭素、
’ 105.4ppmにβ−ソホローヌ部のアノマー炭
素の各シグナルが検出されていることがわかる。また、
試料3とレバウテ゛イオサイドーAの炭素−13核磁気
共鳴ヌベクトルを比較すると、試料3ではレバウディオ
サイド−Aに見られなかった84.6 ppm 、 8
5.6ppm。From the carbon-13 nuclear magnetic resonance Nubek l-/shikara shown in the figure, β-D bound to C-18-OH was present at 98.1 ppm.
-Anomeric carbon of glucone, 96.2 ppmK
(, -19 varnish 1, anomeric carbon of chilled crucose,
It can be seen that each signal of the anomeric carbon of the β-sophorone moiety is detected at 105.4 ppm. Also,
Comparing the carbon-13 nuclear magnetic resonance vectors of sample 3 and rebaudioside-A, sample 3 has 84.6 ppm and 8
5.6ppm.
105、7 ppm 、 106.2 ppm 、 1
06.8 ppmの各シグナルが検出されていた。この
シグナルのうちで105.7ppm、 106.2 p
pm 、 106.8 ppmのシグナルは非常に近接
した位置にあるため帰属できないが、このシグナルのい
ずれかが本発明による反応にて転移したβ−D−グルコ
ースのアノマー炭素のシグナルであると思われる。また
、84.6 ppm、 85.6 ppm1.3−結合
によって、導入を受けた糖の2,3゜4のいずれかの2
個分のシグナルがずれたためであると考えられる。105.7 ppm, 106.2 ppm, 1
Each signal of 0.06.8 ppm was detected. Of this signal, 105.7 ppm, 106.2 p
The signals at 106.8 ppm and 106.8 ppm cannot be assigned because they are located very close to each other, but it is thought that one of these signals is a signal of the anomeric carbon of β-D-glucose transferred in the reaction according to the present invention. . In addition, 84.6 ppm, 85.6 ppm1.3-bonds, 2,3゜4 of the introduced sugar.
This is thought to be due to the individual signals being shifted.
これらの結果を総合して考えると、試料3の化学構造は
、ステビオサイドのG−2の糖残基に、β−1,3−グ
ルコシル結合が導入された新物質であると7考えられる
。Considering these results in total, the chemical structure of sample 3 is considered to be a new substance in which a β-1,3-glucosyl bond is introduced into the G-2 sugar residue of stevioside.
実験4 官能検査
4−1 甘味度の比較
実験2で得た本発明による粉末状甘味料(試料 ・2)
について対照をステビオサイドとして甘味度の比較をし
だ。Experiment 4 Sensory test 4-1 Comparison of sweetness level Powdered sweetener according to the present invention obtained in Experiment 2 (Sample 2)
The sweetness level was compared using stevioside as a control.
試験液として、試料2、ステビオサイドをそれぞれ、1
00■とり、水を加えて200−とじて検査に供した。As test liquids, sample 2 and stevioside were added at 1 volume each.
A sample of 0.00 mm was taken, water was added thereto, the sample was closed with 200 mm, and the sample was subjected to an examination.
官能検査パネルは、甘味の評価にすぐれた検査員5名に
よって行った。The sensory test panel was conducted by five inspectors who were excellent in evaluating sweetness.
その結果
試料2の甘味が強い 1名
ステビオサイドの甘味が強い 4名
となり、試料2の甘味度は、ステビオサイドより弱いと
見なされた。これは、本発明の反応によってステビオサ
イドに等モル以上のD−グルコースがβ−1,3−結合
し、反応生成物の分子量がステビオサイドよシ大きくな
ったためであると考えられた。As a result, 1 person said that sample 2 had a strong sweetness, and 4 people said that stevioside had a strong sweetness, and the sweetness of sample 2 was considered to be weaker than that of stevioside. This was thought to be because the reaction of the present invention resulted in β-1,3-bonding of D-glucose in an amount equal to or more than the same mole to stevioside, and the molecular weight of the reaction product became larger than that of stevioside.
そこで甘味度をそろえるため、試料2の濃度は0.05
%としておき、対照品ヌテビオサイドの濃度を0.04
%、O,Oa%、0.02%になるように調製した各溶
液との比較をしてみた。その結果を表8に示す。Therefore, in order to equalize the sweetness level, the concentration of sample 2 was 0.05.
%, and the concentration of the control product Nutebioside was 0.04.
%, O, Oa%, and each solution prepared to have a concentration of 0.02% was compared. The results are shown in Table 8.
以下余白
表3 甘味度の比較
表3に示した結果から、試料2の0.05%溶液の甘味
度は、ステビオサイドの0035%程度の溶液の甘味度
に等しいことがわかる。Margin Table 3 Below: Comparison of Sweetness Levels From the results shown in Table 3, it can be seen that the sweetness level of the 0.05% solution of Sample 2 is equal to the sweetness level of the approximately 0.035% solution of stevioside.
4−2 甘味質の比較
試験液は甘味度をそろえるため、試料2は0.05%と
し、対照品ステビオサイドは0035%の濃度に調製し
て試験に供した。結果は、
ステビオサイドの甘味が良好 0名
試料2の甘味が良好 5名
となシ、全員一致して試料2が良好な甘味質であるとの
結論を得た。4-2 Comparison of sweetness quality In order to make the test solutions uniform in sweetness, sample 2 was prepared at a concentration of 0.05%, and the control product stevioside was prepared at a concentration of 0.035% and used for the test. The results were as follows: Stevioside had a good sweet taste. 0 people had a good sweet taste. Sample 2 had a good sweet taste. 5 people unanimously concluded that Sample 2 had a good sweet taste.
甘味質が良好である理由は、対照品ステビオサイドは特
有の強い苦味を有し、不快な残株を呈するのに対し、試
料2には苦味が感じられず、不快な残株を呈さないされ
やかでまろやかな甘味であるとされた。The reason why the sweetness quality is good is that the control product stevioside has a unique strong bitter taste and exhibits unpleasant residue, whereas Sample 2 does not have a bitter taste and does not exhibit unpleasant residue. It was said to have a mild and sweet taste.
これらの実験結果から、本発明による甘味料は、ステビ
オサイド特有の苦味や不快な残株を呈さないされやかで
捷ろやかな良質の甘味料であることがわかる。これは、
本発明の反応によってステビオサイドがβ−1,3−グ
リコシルヌテビオサイド類に変換された結果によるもの
と結論される。These experimental results show that the sweetener according to the present invention is a smooth, supple, and high-quality sweetener that does not exhibit the bitter taste or unpleasant residue peculiar to stevioside. this is,
It is concluded that this is due to the conversion of stevioside into β-1,3-glycosyl nutebiosides by the reaction of the present invention.
次に本発明に関する1〜2の実施例について述べる。 Next, first and second embodiments of the present invention will be described.
実施例1
薄層クロマトグラフ分析によってレバウディオサイド−
A以外のヌテビア系甘味成分か検出されなくなるまでに
高度に精製された純レバウディオサイド−Alfを00
1Mリン酸カリウム緩衝液、pH6,0、50++d!
、に加熱溶解し、さらに、酵母グルカン12を加え、酵
素2の0.5−を加えて50℃にて1夜反応させた。反
応液を沸騰水中に10分間保って酵素を加熱失活させ、
10,000 X G 10分間の遠心にて未反応の酵
母グルカンを除去し、液状甘味料を得た。Example 1 Rebaudioside was determined by thin layer chromatography analysis.
Pure rebaudioside-Alf that has been highly purified to the point that no nutevia-based sweet components other than A can be detected.
1M potassium phosphate buffer, pH 6.0, 50++d!
Further, yeast glucan 12 and 0.5-enzyme 2 were added, and the mixture was reacted at 50° C. overnight. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme by heating.
Unreacted yeast glucan was removed by centrifugation at 10,000×G for 10 minutes to obtain a liquid sweetener.
第6図に、この反応前後の薄層クロマトグラムを示す。FIG. 6 shows thin layer chromatograms before and after this reaction.
図面から、本発明による反応にてレバウディオサイド−
Aにも糖転移を生じていることがわかる。From the drawings, it can be seen that rebaudioside is produced in the reaction according to the present invention.
It can be seen that sugar transfer occurs in A as well.
また、反応の前後の溶液について官能検査してみたとこ
ろ、甘味度は反応前後で差が認められなかったが、甘味
質は反応前の、溶液より反応後の溶液のほうがやや良質
であることが認められた。In addition, when we conducted a sensory test on the solutions before and after the reaction, we found that there was no difference in the degree of sweetness before and after the reaction, but the quality of the sweetness was slightly better in the solution after the reaction than in the solution before the reaction. Admitted.
このことから、本発明による方法では、レバウディオサ
イド−Aにも反応が進行し、β−1,3−グリコジルレ
バウディオサイド−A類が生成されその結果、さらにけ
味質が向上することがわかる。From this, in the method according to the present invention, the reaction also progresses to rebaudioside-A, producing β-1,3-glycosyl rebaudioside-A, and as a result, the taste quality is further improved. I understand that.
すなわち、本発明による方法で製造される甘味料は、本
発明を実施しない甘味料に比し、甘味′αの向上も著し
く(特にステビオサイドは、特有の苦味や不快な残株が
消去されるため、本発明の効果は顕著である。一方、レ
バウディオサイド−Aはもともと良質の甘味質を有して
いるが、本発明の実施によってさらに向上させることが
できる。In other words, the sweetener produced by the method according to the present invention has significantly improved sweetness ′α compared to the sweetener that does not carry out the present invention (particularly in the case of stevioside, since the characteristic bitterness and unpleasant residual taste are eliminated) , the effects of the present invention are remarkable. On the other hand, although rebaudioside-A originally has good sweetness, it can be further improved by implementing the present invention.
)、従って、本例のす法科は食品や医薬品の甘味料とし
て特に好ましいものであり、有用に利用されるものであ
ると信する。), Therefore, we believe that the present invention is particularly preferable as a sweetener for foods and medicines, and can be usefully used.
実施例2
市販ステビア抽出精製物(商品名:ステビア5F−AB
、 池田塘化工業株式会社製)2?を0.oIM リ
ン酸カリウム緩衝液5o−に溶解し、パキマン22.酵
素1を2 mll加えて50℃、1夜反応させた。反応
l溶液を沸1俺水中に1o分間保って酵素を加熱失活さ
せ、未反応のパキマン及び不溶物を10.000xG、
10分間の遠心で除去し、液状は法科を得た。Example 2 Commercially available purified stevia extract (product name: Stevia 5F-AB
, manufactured by Ikeda Toka Kogyo Co., Ltd.) 2? 0. oIM Potassium phosphate buffer 5o- dissolved in Pachyman 22. 2 ml of Enzyme 1 was added and reacted at 50°C overnight. The reaction solution was kept in boiling water for 10 minutes to inactivate the enzyme by heating, and unreacted pachyman and insoluble matter were removed at 10.000 x G.
It was removed by centrifugation for 10 minutes, and the liquid was obtained.
第7図に、この反応前後の範層クロマトグラムを示す。FIG. 7 shows a range chromatogram before and after this reaction.
図面から、本発明の反応によって種々の反応生成物が生
じていることがゎがる。すなわち、本発明に用いた酵素
は、ステビオサイドにもレバウディオサイド−Aにも反
応が進行するため、このような種々な反応生成物のスポ
ットか出現したものと考えられる。□
一方、反応前後の溶液について官能検査してみたところ
、本発明の液状甘味料は、反応前の甘味質に見られた苦
味・渋味や不快な残株を呈さないされやかでまろやかな
良質甘味であった。また、反応前後でのH′味度には、
差か認められなかった。From the drawings, it can be seen that various reaction products are produced by the reaction of the present invention. That is, since the enzyme used in the present invention reacts with both stevioside and rebaudioside-A, it is thought that spots of such various reaction products appeared. □ On the other hand, when we conducted a sensory test on the solution before and after the reaction, we found that the liquid sweetener of the present invention has a smooth and mellow taste that does not exhibit the bitterness, astringency, or unpleasant residual taste seen in the sweetness before the reaction. It had a good quality sweetness. In addition, the H' taste level before and after the reaction is as follows:
I couldn't see any difference.
第] 図は、P−ニトロフェニル−β−D −り/L。
コシドとラミナリンとの混合液に酵素2を作用させたも
の(1)と、同酵素を作用させなかった対照品(21の
ペーノゼークロマトグラム。
第2図は、ステビオサイドとラミナリンとの混合溶液に
酵素2を作用させることにより得られた試料1(3)及
び他の標準対照品(1°ステビオールビオサイド、2
ステビオサイド、4:レバウデイオサイドーA、5:レ
パウデイオサイドーB。
6:グルコース)の薄層クロマトグラフ、第3図は、試
料3(2)及びその他の標準対照品(1:ステビオサイ
ド、3 レバウディオサイド−A)の薄層クロマトグラ
ム、第4図は試料3のアルカリ加水分解物(2)及びそ
の他の標準対照品(l:ステビオールビオサイドとレバ
ウディオサイド−B1 ステビオサイドとレバウディオ
サイド−Aとの混合物、3:レバウディオサイドB14
:ステビオ−・ルビオサイド)の薄層クロマトグラム、
第5図は試料3の炭素及核磁気共鳴スペクトル、第6図
はレバウディサイドーAと酵母グルカンとして酵素2を
作用させたもの(3)と、その他標準対照品(1:し、
バラディオサイド−Aと酵母グルカン、2:酵母グルカ
ン+酵素2.4ニゲルコース、5:レバウディオサイド
−A)の薄層クロマトグラム、第7図は、市販ステビア
抽出精製物とバキマンとζこ酵素1を作用させたもの(
3)と、その他の標準対照品(1:パキしンと酵素1,
2:ステビア抽出精製物とパキマン、4:ステビオサイ
ド、5:レバウディオサイド−A16:グルコース)の
薄層クロマトクラムである。The figure shows P-nitrophenyl-β-D-ri/L. Penose chromatograms of a mixed solution of coside and laminarin treated with enzyme 2 (1) and a control product (21) without the same enzyme. Figure 2 shows a mixed solution of stevioside and laminarin. Sample 1 (3) obtained by treating enzyme 2 with other standard controls (1° steviol bioside, 2
Stevioside, 4: Rebaudioside-A, 5: Rebaudioside-B. 6: Glucose), Figure 3 is the thin layer chromatogram of Sample 3 (2) and other standard controls (1: Stevioside, 3 Rebaudioside-A), Figure 4 is the sample Alkaline hydrolyzate (2) of 3 and other standard reference products (l: mixture of steviolbioside and rebaudioside-B1, stevioside and rebaudioside-A, 3: rebaudioside B14
: Thin layer chromatogram of Stevio-Rubioside),
Figure 5 shows the carbon and nuclear magnetic resonance spectra of Sample 3, Figure 6 shows the spectra of rebaudicide A and yeast glucan treated with Enzyme 2 (3), and other standard controls (1:
Thin layer chromatogram of Valadioside-A and Yeast Glucan, 2: Yeast Glucan + Enzyme 2.4 Nigelcose, 5: Rebaudioside-A), Figure 7 shows commercially available purified Stevia extract, Bakiman and ζ This enzyme 1 is applied (
3) and other standard controls (1: paxin and enzyme 1,
2: Stevia extract purified product and Pachyman, 4: Stevioside, 5: Rebaudioside-A16: Glucose).
Claims (3)
メークリコシルレバウデイオサイド−AMを含−4,t
−ることを特徴とする甘味料。(1) -4,t containing β-glycosylstehioside and/or make glycosyl rebaudioside-AM
- A sweetener characterized by:
ド−八を含有する特許請求の範囲第(11118i記!
lνの甘味料。(2) Claim No. 11118i containing stevioside and/or rebaudioside-8!
lν sweetener.
ド−Aに、θ−グルコシル糖化合物を基質として、θ−
クルコシル転移酵素を作用させて生成するθ−グリコジ
ルステビオサイド峯自及び/又はβ−グリコジルレバウ
ディオサイド−A Yiを含有することを特徴とする甘
味料の製造法。(3) Using stevioside and/or rebaudioside-A with a θ-glucosyl sugar compound as a substrate, θ-
1. A method for producing a sweetener comprising θ-glycodyl stevioside Mineji and/or β-glycodyl rebaudioside-A Yi produced by the action of curcosyltransferase.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57229417A JPS59120073A (en) | 1982-12-28 | 1982-12-28 | Sweetener and its preparation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57229417A JPS59120073A (en) | 1982-12-28 | 1982-12-28 | Sweetener and its preparation |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS59120073A true JPS59120073A (en) | 1984-07-11 |
Family
ID=16891897
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP57229417A Pending JPS59120073A (en) | 1982-12-28 | 1982-12-28 | Sweetener and its preparation |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS59120073A (en) |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6291161A (en) * | 1985-10-16 | 1987-04-25 | Dainippon Ink & Chem Inc | Stevia sweetener and production thereof |
KR20040026747A (en) * | 2002-09-26 | 2004-04-01 | 바이오스펙트럼 주식회사 | Method for preparation of rebaudioside A using microorganism. |
KR100851610B1 (en) * | 2001-09-21 | 2008-08-12 | 디아이씨 가부시끼가이샤 | Sweetener and process for producing the same |
US8030481B2 (en) | 2007-05-21 | 2011-10-04 | The Coca-Cola Company | Stevioside polymorphic and amorphous forms, methods for their formulation, and uses |
EP2482676A2 (en) * | 2009-06-16 | 2012-08-08 | Zhejiang Green World Bio-Tech Engineering Co., Ltd. | Composition comprising rebaudioside d fore reducing or eliminating aftertaste and preparation method thereof |
US8791253B2 (en) | 2006-06-19 | 2014-07-29 | The Coca-Cola Company | Rebaudioside A composition and method for purifying rebaudioside A |
US9012626B2 (en) | 2006-06-19 | 2015-04-21 | The Coca-Cola Company | Rebaudioside a composition and method for purifying rebaudioside a |
US9402411B2 (en) | 2011-01-28 | 2016-08-02 | Tate & Lyle Ingredients Americas Llc | Stevia blends containing rebaudioside B |
CN106071615A (en) * | 2016-06-08 | 2016-11-09 | 郑书旺 | A kind of fruity stevioside solid beverage |
US9578895B2 (en) | 2010-08-23 | 2017-02-28 | Epc (Beijing) Natural Products Co., Ltd. | Rebaudioside A and stevioside compositions |
US9609887B2 (en) | 2012-08-01 | 2017-04-04 | Tate & Lyle Ingredients Americas Llc | Sweetener compositions containing monk fruit extract and rebaudiosides A and B |
US9795156B2 (en) | 2011-03-17 | 2017-10-24 | E.P.C (Beijing) Plant Pharmaceutical Technology Co., Ltd | Rebaudioside B and derivatives |
US10264811B2 (en) | 2014-05-19 | 2019-04-23 | Epc Natural Products Co., Ltd. | Stevia sweetener with improved solubility |
US10357052B2 (en) | 2014-06-16 | 2019-07-23 | Sweet Green Fields USA LLC | Rebaudioside A and stevioside with improved solubilities |
US10485256B2 (en) | 2014-06-20 | 2019-11-26 | Sweet Green Fields International Co., Limited | Stevia sweetener with improved solubility with a cyclodextrin |
-
1982
- 1982-12-28 JP JP57229417A patent/JPS59120073A/en active Pending
Cited By (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6291161A (en) * | 1985-10-16 | 1987-04-25 | Dainippon Ink & Chem Inc | Stevia sweetener and production thereof |
KR100851610B1 (en) * | 2001-09-21 | 2008-08-12 | 디아이씨 가부시끼가이샤 | Sweetener and process for producing the same |
KR20040026747A (en) * | 2002-09-26 | 2004-04-01 | 바이오스펙트럼 주식회사 | Method for preparation of rebaudioside A using microorganism. |
US8791253B2 (en) | 2006-06-19 | 2014-07-29 | The Coca-Cola Company | Rebaudioside A composition and method for purifying rebaudioside A |
US9012626B2 (en) | 2006-06-19 | 2015-04-21 | The Coca-Cola Company | Rebaudioside a composition and method for purifying rebaudioside a |
US8030481B2 (en) | 2007-05-21 | 2011-10-04 | The Coca-Cola Company | Stevioside polymorphic and amorphous forms, methods for their formulation, and uses |
US9131718B2 (en) | 2009-06-16 | 2015-09-15 | Epc (Beijing) Natural Products Co., Ltd. | Process for rebaudioside D |
US9635878B2 (en) | 2009-06-16 | 2017-05-02 | Epc (Beijing) Natural Products Co., Ltd. | Process for rebaudioside D |
CN104286779A (en) * | 2009-06-16 | 2015-01-21 | 伊比西(北京)植物药物技术有限公司 | Composition comprising rebaudioside D and application thereof |
EP2482676A4 (en) * | 2009-06-16 | 2014-07-09 | Zhejiang Green World Bio Tech Engineering Co Ltd | Composition comprising rebaudioside d fore reducing or eliminating aftertaste and preparation method thereof |
EP2826386A1 (en) * | 2009-06-16 | 2015-01-21 | EPC (Beijing) Natural Products Co., Ltd. | Stevia composition comprising rebaudioside D |
EP2482676A2 (en) * | 2009-06-16 | 2012-08-08 | Zhejiang Green World Bio-Tech Engineering Co., Ltd. | Composition comprising rebaudioside d fore reducing or eliminating aftertaste and preparation method thereof |
US11202462B2 (en) | 2010-08-23 | 2021-12-21 | Sweet Green Fields International Co., Limited | Rebaudioside A and stevioside compositions |
US10285425B2 (en) | 2010-08-23 | 2019-05-14 | Epc Natural Products Co. Ltd | Rebaudioside A and stevioside compositions |
US9578895B2 (en) | 2010-08-23 | 2017-02-28 | Epc (Beijing) Natural Products Co., Ltd. | Rebaudioside A and stevioside compositions |
US10583314B2 (en) | 2011-01-28 | 2020-03-10 | Tate & Lyle Ingredients Americas Llc | Stevia blends containing rebaudioside B |
US11801402B2 (en) | 2011-01-28 | 2023-10-31 | Tate & Lyle Solutions Usa Llc | Stevia blends containing rebaudioside b |
US9402411B2 (en) | 2011-01-28 | 2016-08-02 | Tate & Lyle Ingredients Americas Llc | Stevia blends containing rebaudioside B |
US9795156B2 (en) | 2011-03-17 | 2017-10-24 | E.P.C (Beijing) Plant Pharmaceutical Technology Co., Ltd | Rebaudioside B and derivatives |
US11510428B2 (en) | 2011-03-17 | 2022-11-29 | Sweet Green Fields International Co., Limited | Rebaudioside B and derivatives |
US9609887B2 (en) | 2012-08-01 | 2017-04-04 | Tate & Lyle Ingredients Americas Llc | Sweetener compositions containing monk fruit extract and rebaudiosides A and B |
US10561165B2 (en) | 2014-05-19 | 2020-02-18 | Sweet Green Fields International Co., Limited | Stevia sweetener with improved solubility |
US11206857B2 (en) | 2014-05-19 | 2021-12-28 | Sweet Green Fields International Co., Limited | Stevia sweetener with improved solubility |
US10264811B2 (en) | 2014-05-19 | 2019-04-23 | Epc Natural Products Co., Ltd. | Stevia sweetener with improved solubility |
US10568351B2 (en) | 2014-06-16 | 2020-02-25 | Sweet Green Fields USA LLC | Rebaudioside A and stevioside with improved solubilities |
US10357052B2 (en) | 2014-06-16 | 2019-07-23 | Sweet Green Fields USA LLC | Rebaudioside A and stevioside with improved solubilities |
US11241031B2 (en) | 2014-06-16 | 2022-02-08 | Sweet Green Fields Usa, Llc | Rebaudioside A and stevioside with improved solubilities |
US10485256B2 (en) | 2014-06-20 | 2019-11-26 | Sweet Green Fields International Co., Limited | Stevia sweetener with improved solubility with a cyclodextrin |
CN106071615A (en) * | 2016-06-08 | 2016-11-09 | 郑书旺 | A kind of fruity stevioside solid beverage |
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