CN105838759A - Preparation method of stevia sugar derivative - Google Patents
Preparation method of stevia sugar derivative Download PDFInfo
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- CN105838759A CN105838759A CN201610181707.6A CN201610181707A CN105838759A CN 105838759 A CN105838759 A CN 105838759A CN 201610181707 A CN201610181707 A CN 201610181707A CN 105838759 A CN105838759 A CN 105838759A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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Abstract
The invention relates to a preparation method of a stevia sugar derivative and belongs to the technical field of food chemical engineering. With a stevia sugar derivative compound (I) as a substrate, a reaction is carried out under the catalysis of an UDP glucosyl transferase to the substrate to generate a stevia sugar derivative compound (IV) in the presence of a glucosyl group donor. The preparation method is green and safe, is low in production cost and short in period, greatly improves competitiveness of the product, is simple in operation, is high in conversion rate and is simple in purification. The product is high in purity, can be used in food and beverage industries, and has greatly application value.
Description
Technical field
The present invention relates to the preparation method of a kind of stevioside derivant, belong to food chemistry technical field.
Background technology
Stevioside is a kind of new type natural sweeting agent extracted the leaf from catananche's Folium Stevlae Rebaudianae, stem, be a kind of natural,
Green, the functional food of health care, it has fragrance, refrigerant taste, also has the feature that sugariness is high, heat energy is low, its sugariness
It is 200-300 times of sucrose, and calorific value is only sucrose volume 1/300, be the ideal sweeting agent of a kind of alternative sucrose.Along with
The mankind are known as, by Food Science man, the sweeting agent that future world is most with prospects to healthy, green attention, stevioside.
Stevioside is considered the good supplementary of the mankind and health care medicine by international medical community.Prove through a large amount of scientific experimentss,
Stevioside is conducive to regulation blood glucose and blood pressure, the most edible regulating blood sugar level, and prophylaxis of hypertension improves hypostension；
Stevioside also has some bacterial growth of suppression and breeding and the effect stoping it to infect, it is possible to treatment flu and influenza and pre-preventing decayed tooth
The diseases such as tooth；Stevioside can reduce people's demand to sugar with fat, regulates its diet, reduces people's demand to tobacco and wine,
Prevent the generation of the diseases such as diabetes, obesity, heart disease；Meanwhile, stevioside also has skin repair and improving effect,
It can treat dermatosis, smoothes away wrinkles, and removes cicatrix etc..
Stevioside can be widely applied to the industries such as food, beverage, flavouring agent, medicine, daily-use chemical industry, wine brewing, cosmetics, relatively
Use sucrose escapable cost about 60%, and be of high nutritive value.The stability of stevioside, metabolic pathway and safety thereof are by deeply
Entering research, its China's Ministry of Public Health, Ministry of Light Industry's approval uses, and is also recognized as by U.S. food Drug Administration
The rank of " GRAS (being commonly considered as safe) ".Stevioside is as the natural substitute of sucrose, and it has a extensive future.
Though stevioside has numerous advantage, but its serious bitter aftertaste, hinder its extensive application.At present, Flos Chrysanthemi
The improvement of sugar mouthfeel can be realized by enzyme process bioconversion.Have been reported, it is possible to use cyclodextrin glycosyl transferases modification Flos Chrysanthemi
Sugar, to improve its mouthfeel and taste matter, but the position of this enzyme transfer glycosyl is the most single-minded, and product is impure, and its sugariness the most seriously drops
Low.
The double glycosides of Flos Chrysanthemi is converted into the reaction etc. of rebaudioside A and D to stevioside and the conversion of RB and stevioside.This
Study for numerous components of stevioside a bit, the most very little, and also its modified mouthfeel and taste matter are also Pangs
Big research.Seek efficient metabolizing enzymes, conversion process and Novel sweet inulin derivant and be still the research that stevioside is following
Trend.
Summary of the invention
Present invention solves the technical problem that and be: propose a kind of cost that can be relatively low and the Flos Chrysanthemi of shorter production cycle output high-quality
The preparation method of sugar derivatives, the method with compound (I) as raw material, under the catalytic action of UDPG based transferase
Reaction generates stevioside derivative compound (IV).
In order to solve above-mentioned technical problem, the technical scheme that the present invention proposes is: the preparation method of a kind of compound (IV),
With compound (I) as substrate, make described substrate in the presence of glucosyl group donor, at UDPG based transferase
Reacting generating compound (IV) under catalytic action, described preparation method is as follows:
Preferably, described UDPG based transferase is carried by fermenting and producing portion of Nanjing Nuoyun Biological Technology Co., Ltd.
Supply.
Preferably, the consumption of described UDPG is 0.0135～5.41g/L.
Preferably, in microbial hosts express UDPG based transferase, described microbial hosts selected from escherichia coli,
Bacillus, yeast, aspergillosis or Pichia sp..
Preferably, described reaction is at MgCl2Concentration is 0～0.286g/L, and temperature is 20～37 DEG C, the water of pH5.0～9.0
Carrying out in phase system, the response time is 0.5～72h.
Preferably, described reaction is carried out in the Tris-HCl buffer of pH8.0.
Preferably, substrate initial concentration is 0.676～54.05g/L.
Preferably, take UDPG based transferase 444mg, use sterilized water re-suspended cell, and ultrasonic disruption is thin in ice bath
Born of the same parents, are the crude enzyme liquid used by reaction；Precision weighs sample, is configured to 1.85ml system, and wherein the end of compound (I) is dense
Degree 2.0g/L, UDPG are 2.70g/L, and add the MgCl of 0.286g/L2；It is subsequently adding crude enzyme liquid, and adds Tris-HCl
PH of buffer 8.0 to system is 1.85ml, initial action；25 DEG C of constant-temperature tables vibrate 38h with 150rpm, and 100 DEG C are boiled end
Only reaction, 13000g is centrifuged 10min, takes supernatant as sample, utilizes macroporous resin to carry out sample just and purify, and use LC-MS
The purity of the compound that method detection purifies.
Beneficial effect:
The present invention with stevioside derivative compound (I) as substrate, make described substrate in the presence of glucosyl group donor, at UDP-
Under the catalytic action of glucosyltransferase, reaction generates stevioside derivative compound (IV), production technology green safety, and life
It is short to produce low cost, cycle, improves the competitiveness of product greatly.Owing to the present invention is easy and simple to handle, transformation efficiency is high, purifies and holds
Easily, products obtained therefrom purity is high, can be used for bag and bottle industry, has important using value.
Accompanying drawing explanation
Below in conjunction with the accompanying drawings the present invention is described further.
Fig. 1 is the HPLC spectrogram of compound (IV)
Fig. 2 is the MS spectrogram of compound (IV)
Detailed description of the invention
The UDPG based transferase (i.e. uridine diphosphoglucose based transferase) of the present invention is had by Nanjing promise cloud biotechnology
Fermenting and producing portion of limit company provides, and below UDPG based transferase is called for short M303.
Embodiment 1:
The structure of recombination bacillus coli and abduction delivering
Utilize molecular biology and technique for gene engineering etc. to obtain the expression of recombinant e. coli bacterial strain containing genes of interest, then will
Recombination bacillus coli fermentation culture, the abduction delivering preparation reconstitution cell containing destination protein.It specifically comprises the following steps that
1) primer segments needed for synthesis, the UDPG based transferase M303 needed for being obtained by PCR amplification is encoded
DNA fragmentation, and by homologous recombination technique, be incorporated in the expression cassette of pNYK expression vector.
2) by recombinant plasmid transformed to escherichia coli, it is thus achieved that the engineering bacteria J303 containing genes of interest.
3) being placed in TB culture medium by 1ml engineering bacteria J303,250rpm, 37 DEG C of shaken cultivation, to OD600=1.0, add
Final concentration of 0.1mM IPTG is in 25 DEG C of shaken cultivation 16h.Induce after terminating with 10000g, 5min centrifugal collecting cell, put
Store for future use in-80 DEG C.Obtain the thalline containing M303 albumen.
Embodiment 2:
Utilizing compound (I) is that substrate prepares compound (IV)
Take wet thallus M303 444mg, use sterilized water re-suspended cell, and ultrasonic disruption cell in ice bath, be reaction institute
Crude enzyme liquid.Precision weighs sample, is configured to 1.85ml system, wherein substrate compounds (I) final concentration 2.0g/L,
UDPG is 2.70g/L, and adds the MgCl of 0.286g/L2；It is subsequently adding crude enzyme liquid, and adds final concentration 0.1M's
Tris-HCl buffer (pH8.0) to system is 1.85ml, initial action.25 DEG C of constant-temperature tables vibrate 38h with 150rpm,
100 DEG C are boiled termination reaction.13000g is centrifuged 10min, takes supernatant as sample, utilizes macroporous resin to carry out sample just and purifies,
And use the purity of the compound (IV) of LC-MS method detection purification.
Embodiment 3:
The determination of best metal ion concentration
Thalline M303 444g in Example 1, according to the method in embodiment 2, is transferred in 5ml centrifuge tube, adds
The compound (I) of final concentration of 2.0g/L, UDPG is 2.70g/L and MgCl2, and it is slow to add 0.1M Tris-HCl
Rush liquid (pH8.0).As stated above, take Duplicate Samples, add MgCl2Final concentration be respectively 0,0.1,0.286g/L, instead
After answering 38h, take after sample boils termination centrifugal, Supernatant samples is carried out HPLC analysis.Wherein MgCl2When being 0.286,
Compound (IV) yield is the highest.·
Embodiment 4:
The determination of optimal reaction temperature
Thalline M303 444g in Example 1, according to the method in embodiment 2, is transferred in 5ml centrifuge tube, adds
The Tris-HCl buffer (pH8.0) of final concentration of 0.1M, compound (I) is 2.0g/L, and UDPG is 2.70g/L.
Taking Duplicate Samples as stated above, respectively at 20,25,30,37 DEG C, 150rpm carries out catalytic reaction, after reaction 30h, and sampling
Product are centrifugal after boiling termination, and Supernatant samples is carried out HPLC analysis.Wherein temperature is 25 DEG C, and compound (IV) yield is the highest.
Embodiment 5:
The determination of optimum response pH
Thalline M303 444g in Example 1, according to the method in embodiment 2, is transferred in 5ml centrifuge tube, adds
Entering the Tris-HCl buffer of final concentration of 0.1M, compound (I) is 2.0g/L, and UDPG is 2.70g/L.By upper
The method of stating takes Duplicate Samples, and wherein pH is adjusted to 5.0,6.0,7.0,7.5,8.0,9.0 respectively.In 30 DEG C, 150rpm urges
Change reaction, after reaction 38h, take sample and be centrifuged, Supernatant samples is carried out HPLC analysis.When wherein pH is 8.0, compound
(IV) yield is the highest.
Embodiment 6:
Optimal substrate addition
Thalline M303 444g in Example 1, according to the method in embodiment 2, is transferred in 5ml centrifuge tube, adds
The Tris-HCl buffer (pH8.0) of final concentration of 0.1M, UDPG is 2.70g/L.As stated above, take Duplicate Samples,
And be separately added into final concentration of 0.676,0.901,1.35,2.0,5.41,13.51,27.03, the compound (I) of 54.05g/L.
After reaction 38h, take after sample boils termination centrifugal, Supernatant samples is carried out HPLC analysis.When wherein adding 2.0g/L substrate,
The yield of compound (IV) is the highest.
Embodiment 7:
Compound (IV) structural analysis
The method that the structure of compound (I) has been analyzed by carrying out hydrogen, carbon and ID NMR speetna resolves.Compound (I)
Molecular weight (1127) is comparable to steviol and adds five glucoses, according to excess M303 enzyme exist characteristic i.e. in β-1,2 glycosidic bonds
Add two glucose groups continuously and infer compound (IV) C62H100O38(1451.8) structure, and record molecule with LC
Amount (1451) is confirmed.Compound (IV) molecular weight is comparable to steviol and adds 7 glucoses.
Above-described embodiment is only for technology design and the feature thereof of the explanation present invention, it is intended to those skilled in the art can easily be managed
Solve present disclosure and implement according to this, can not limit the scope of the invention with this.In every case real according to spirit of the present invention
The equivalence that matter is done changes or modifies, and all should contain within protection scope of the present invention.
Claims (8)
1. the preparation method of a compound (IV), it is characterised in that: with compound (I) as substrate, make the described end
Thing in the presence of glucosyl group donor, reacting generating compound (IV) under the catalytic action of UDPG based transferase,
Described preparation method is as follows:
The preparation method of compound the most according to claim 1 (IV), it is characterised in that: described UDP-Portugal
Glucosyl transferase is provided by fermenting and producing portion of Nanjing Nuoyun Biological Technology Co., Ltd..
The preparation method of compound the most according to claim 1 (IV), it is characterised in that: described UDP-Portugal
The consumption of grape sugar is 0.0135～5.41g/L.
The preparation method of compound the most according to claim 1 (IV), it is characterised in that: in microbial hosts
Express UDPG based transferase, described microbial hosts selected from escherichia coli, bacillus, yeast, aspergillosis or
Pichia sp..
The preparation method of compound the most according to claim 1 (IV), it is characterised in that: described reaction is at MgCl2
Concentration is 0～0.286g/L, and temperature is 20～37 DEG C, carries out in the aqueous phase system of pH5.0～9.0, the response time be 0.5～
72h。
The preparation method of compound the most according to claim 5 (IV), it is characterised in that: described reaction is at pH8.0
Tris-HCl buffer in carry out.
The preparation method of compound the most according to claim 1 (IV), it is characterised in that: substrate initial concentration is
0.676～54.05g/L.
The preparation method of compound the most according to claim 7 (IV), it is characterised in that: take UDPG base and turn
Move enzyme 444mg, use sterilized water re-suspended cell, and ultrasonic disruption cell in ice bath, be the crude enzyme liquid used by reaction；Essence
Close weighing sample, be configured to 1.85ml system, wherein the final concentration 2.0g/L of compound (I), UDPG are 2.70g/L,
And add the MgCl of 0.286g/L2；It is subsequently adding crude enzyme liquid, and to add Tris-HCl pH of buffer 8.0 to system is 1.85ml,
Initial action；25 DEG C of constant-temperature tables vibrate 38h with 150rpm, and 100 DEG C are boiled termination reaction, and 13000g is centrifuged 10min, takes
Supernatant, as sample, utilizes macroporous resin to carry out sample just and purifies, and uses the pure of compound that the detection of LC-MS method purifies
Degree.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110656150A (en) * | 2019-10-30 | 2020-01-07 | 山东三元生物科技股份有限公司 | Preparation method of rebaudioside E, and product and application thereof |
| WO2020112957A1 (en) * | 2018-11-27 | 2020-06-04 | Purecircle Usa Inc. | High-purity steviol glycosides |
| WO2020168312A1 (en) * | 2019-02-15 | 2020-08-20 | Purecircle Usa Inc. | High-purity steviol glycosides |
| CN112512337A (en) * | 2018-03-16 | 2021-03-16 | 谱赛科美国股份有限公司 | High purity steviol glycosides |
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| CN105348337A (en) * | 2015-11-19 | 2016-02-24 | 南京诺云生物科技有限公司 | Stevioside derivative prepared by stervioside biotransformation, preparation method and application thereof |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112512337A (en) * | 2018-03-16 | 2021-03-16 | 谱赛科美国股份有限公司 | High purity steviol glycosides |
| JP2021518369A (en) * | 2018-03-16 | 2021-08-02 | ピュアサークル ユーエスエー インコーポレイテッド | High-purity steviol glycoside |
| EP3764815A4 (en) * | 2018-03-16 | 2022-01-26 | PureCircle USA Inc. | High-purity steviol glycosides |
| JP2024109659A (en) * | 2018-03-16 | 2024-08-14 | ピュアサークル ユーエスエー インコーポレイテッド | Highly pure steviol glycosides |
| EP3887383A4 (en) * | 2018-11-27 | 2022-11-09 | PureCircle USA Inc. | High-purity steviol glycosides |
| WO2020112957A1 (en) * | 2018-11-27 | 2020-06-04 | Purecircle Usa Inc. | High-purity steviol glycosides |
| JP7634477B2 (en) | 2018-11-27 | 2025-02-21 | ピュアサークル ユーエスエー インコーポレイテッド | High Purity Steviol Glycosides |
| CN113227111A (en) * | 2018-11-27 | 2021-08-06 | 谱赛科美国股份有限公司 | High-purity steviol glycosides |
| AU2019389030B2 (en) * | 2018-11-27 | 2025-01-02 | Purecircle Usa Inc. | High-purity steviol glycosides |
| JP2022513616A (en) * | 2018-11-27 | 2022-02-09 | ピュアサークル ユーエスエー インコーポレイテッド | High-purity steviol glycoside |
| CN113508124A (en) * | 2019-02-15 | 2021-10-15 | 谱赛科美国股份有限公司 | High-purity steviol glycosides |
| JP2022520257A (en) * | 2019-02-15 | 2022-03-29 | ピュアサークル ユーエスエー インコーポレイテッド | High-purity steviol glycoside |
| WO2020168312A1 (en) * | 2019-02-15 | 2020-08-20 | Purecircle Usa Inc. | High-purity steviol glycosides |
| US12378590B2 (en) | 2019-02-15 | 2025-08-05 | Purecircle Usa Inc. | High-purity steviol glycosides |
| CN110656150A (en) * | 2019-10-30 | 2020-01-07 | 山东三元生物科技股份有限公司 | Preparation method of rebaudioside E, and product and application thereof |
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