CN101974584B - Two-enzyme coupled method for preparing difructose anhydride III - Google Patents
Two-enzyme coupled method for preparing difructose anhydride III Download PDFInfo
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Abstract
The invention discloses a two-enzyme coupled method for preparing difructose anhydride III and belongs to the technical field of functional food bioprocessing. In the method, sucrose is taken as base solution; and sucrose and fructose transferase and synanthrin and fructose transferase are adopted so as to synthesize the difructose anhydride III through a two-enzyme coupled catalytic reaction. The method comprises the following steps of: controlling the concentration of the sucrose substrate to be 20 to 300g/L; reacting the sucrose and fructose transferase, adding the substrate in an amount of 1-20U/g, and carrying out a catalytic reaction at the temperature of between 30 and 55 DEG C for 4 to 12 hours; and adding the synanthrin and fructose transferase, adding the substrate in an amount of 2-15U/g, and carrying out a catalytic reaction at the temperature of between 40 and 60 DEG C for 12 to 36 hours, wherein the biological conversion rate is over 35 percent. The prepared difructose anhydride III is a novel low-calorie sweetener.
Description
Technical field
The present invention relates to a kind of difructose anhydride III biological preparation method, refer in particular to a kind of double enzyme coupling preparation of difructose anhydride III, belong to functional foodstuff biological processing technical field.
Background technology
In recent years, functional food becomes the focus that consumers in general pay close attention to, and its exploitation is the focus of vast food practitioner research and development especially.Diabetes, obesity and cardiovascular disorder crowd's year by year increase and becoming younger is so that focus low in calories, that functional sweetener that have the greater functionality nutritive property becomes concern.
Difructose anhydride III (Difrucose anhydride, DFAIII) be a kind of irreducibility disaccharide of minimum of two fructose combinations, contain a small amount of difructose anhydride III in the plant such as witloof, jerusalem artichoke, in the course of processing of honey, coffee etc., produce a small amount of difructose anhydride III.Its relative sweetness is half of sucrose, and caloric value only has 1/15 of sucrose; Character is very stable, and is non-hygroscopic under 74% relative humidity; Very stable to heat and acid, under the condition of high acidity and high temperature, can not be hydrolyzed, under the normal food processing conditions, hardly brown stain or decomposing phenomenon can appear.Difructose anhydride III also has good physiological function, as not absorbing at digestive tube, and generate energy not, can be used as a kind of sweeting agent; Increase the divergent factor as a kind of, can promote the growth of enteric microorganism, obviously improve defecation, diuresis function; As the functional carbohydrate that promotes that human body absorbs mineral elements such as Ca, Mg, Zn, Cu, promote the growth of bone; Acid is not produced in the Streptococcus oralis utilization that can not be induced to decay tooth, and has the function of anti-dental caries.These advantages hold out broad prospects its application in foodstuffs industry.
Natural difructose anhydride III content seldom extracts separation costs high, and chemosynthesis has many disadvantageous factors, such as separation and purification complexity and contaminate environment; And the Biological preparation difructose anhydride III belongs to natural product, meets human consumer's psychology, therefore adopts biotransformation method to become the focus of difructose anhydride III preparation research.The research of biotransformation method has the history in more than 30 year, Uchiyama T had found inulin ftructotransferase (inulin fructotransferase first from produce the urea Arthrobacter in 1973, EC2.4.1.93), up to now, found that more than ten kind microorganisms can produce this enzyme, mainly concentrate on genus arthrobacter, comprise Arthrobacter urefaciens7116, Arthrobacter sp.A-6, Arthrobacter ilicis OK17B, Arthrobacter globiformisC11-1, Arthrobacter sp.H65-7, Arthrobacter pascens T13-2, Arthrobacter sp.Buo141, Arthrobacter sp.L68-1, in three kinds of other Pseudomonas, also found this enzyme in addition, such as Flavobacterium sp.LC-413, Bacillus sp.Snu-7, Leifsonia sp.T88-4.
The inventor has investigated and has studied prior art further and the method for various product difructose anhydride IIIs has been studied, and proposes to produce the method for difructose anhydride III with a kind of new substrate sucrose and two kinds of enzymes (sucrose fructose-transferring enzyme and inulin ftructotransferase) keying action.Be oligofructose by the sucrose fructose-transferring enzyme with sucrose inversion, add again inulin ftructotransferase, obtain difructose anhydride III.Based on above-mentioned discovery the present invention has been proposed.
Summary of the invention
The double enzyme coupling preparation that the purpose of this invention is to provide a kind of difructose anhydride III of cheapness.Utilize two enzyme coupled methods, the production method of the existing most of single enzymes of difference.
Technical scheme of the present invention:
A kind of double enzyme coupling preparation of difructose anhydride III the steps include:
A, sucrose is dissolved in the deionized water as substrate, is made into the sucrose reaction substrate, sucrose reaction substrate concentration is controlled at 20～300g/L, regulate pH value 5～7, be warming up to 30～55 ℃, add the sucrose fructose-transferring enzyme, add-on is 1～20U/g sucrose, isothermal reaction 4～12 hours;
B, in step (A) afterwards adds inulin ftructotransferase again, and add-on is 2～15U/g sucrose, regulates pH value 5～7, utilizes pair enzyme coupling catalytic reactions technology, 40～60 ℃ of insulation reaction 12～36 hours, and transformation efficiency reaches more than 35%;
C, with step (B) gained converted product solution heating, the enzyme that goes out is lived, and adds the gac of gained system quality mark 1%～3%, decolorization filtering; After being concentrated into a small amount of crystal and separating out, add the ethanol of 2～5 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling; After the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the difructose anhydride III crude product after the drying;
D, recrystallization: the difructose anhydride III crude product that the obtains concentration by massfraction 10%～30% is dissolved in the deionized water, be warming up to 70～80 ℃, the gac that adds difructose anhydride III crude product massfraction 3%, decolorization filtering after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2～5 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling, after the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the difructose anhydride III finished product after the drying.
Requirement according to techniques such as present method transformation efficiency and subsequent extracted, processing, effectively control the concentration of enzyme in conversion fluid, not only can improve transformation efficiency, also can take full advantage of enzyme activity, be that reaction system reaches best stable condition, guarantee the complete processing requirement of product.
The double enzyme coupling preparation of above-mentioned difructose anhydride III, as preferably, in the described steps A, the conversion fluid concentration of substrate is controlled at 20～300g/L.The control of concentration of substrate determines the transformation efficiency of reaction substrate, meet the requirement of subsequent machining technology, therefore effectively control the concentration of reaction substrate, not only can improve reaction conversion ratio, also can utilize fully enzyme activity, make reaction conditions meet the requirement of reaction kinetics, thereby reach stabilization process, enhance productivity save energy.
The double enzyme coupling preparation of above-mentioned difructose anhydride III, as preferably, among the described step B, the amount that adds inulin ftructotransferase in the solution is 2～15U/g (enzyme activity/substrate quality).
The double enzyme coupling preparation of above-mentioned difructose anhydride III, as preferably, described step C and D are treating process.In the production process, disposable products out are difficult to directly reach qualified, therefore need a treating process.
Detect the content of DFAIII with high performance liquid chromatography.HPLC testing conditions: sugared post (Waters Sugur-Pak
TM1,6.5 * 300mm); Moving phase: ultrapure water; Differential refraction detector (Shodex RI101); Column temperature: 80 ℃; Flow velocity: 0.4mL/min; Sample size: 10 μ L.
The ratio of the peak area of the peak area of the DFAIII of detection reaction liquid and certain density standard model DFAIII draws the content of DFAIII in the reaction solution.
Beneficial effect of the present invention:
The transformation efficiency of the inventive method can surpass 35%.
The present invention utilizes the production by biological enzyme, and enzyme activity is high, easily obtains enzyme liquid.The microorganism that produces enzyme all derives from nature, and stability is high, and security is good;
Characteristics of the present invention are the advantages such as technology controlling and process is stable, and production cost is low, and energy consumption is low, and products obtained therefrom is safe and reliable, have the prospect of industrialization.
Description of drawings
Fig. 1 high performance liquid chromatography detects the spirogram that contains of DFAIII
Embodiment
Embodiment 1:
100g sucrose dry product is dissolved in the 1L deionized water, with 1mol/L salt acid for adjusting pH to 5.5, is warming up to 30 ℃, adding sucrose fructose-transferring enzyme is 1U/g to the amount of substrate, and constant temperature transforms 12 hours; Adding inulin ftructotransferase is 15U/g to the amount of substrate again, and 40 ℃ of temperature, pH5.5 reacted 36 hours, and transformation efficiency can reach 37%.The enzyme that goes out is lived, and adds activated carbon decolorizing and filters, and after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2 times of concentrated solution volumes in the concentrated solution, crystallisation by cooling.After the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the DFAIII crude product after the drying.
The DFAIII crude product that obtains is got 100g to be dissolved in the deionized water by the concentration of massfraction 10%, be warming up to 70 ℃, the gac that adds crude product massfraction 3%, decolorization filtering after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling, after the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the DFAIII finished product after the drying.
Detect the content of DFAIII with high performance liquid chromatography, content is seen Fig. 1.HPLC testing conditions: sugared post (Waters Sugur-Pak
TM1,6.5 * 300mm); Moving phase, ultrapure water; Differential refraction detector (ShodexRI101); Column temperature, 80 ℃; Flow velocity, 0.4mL/min; Sample size, 10 μ L.
The ratio of the peak area of the peak area of the DFAIII of detection reaction liquid and certain density standard model DFAIII draws the content of DFAIII in the reaction solution.
Case study on implementation 2:
20g sucrose dry product is dissolved in the 1L deionized water, with 1mol/L salt acid for adjusting pH to 6, is warming up to 55 ℃, adding sucrose fructose-transferring enzyme is 20U/g to the amount of substrate, puts into 55 ℃ of constant temperature and transforms 4 hours; Adding inulin ftructotransferase is 10U/g to the amount of substrate again, and 55 ℃ of temperature, pH6 reacted 12 hours, and transformation efficiency can reach 45%.The enzyme that goes out is lived, and adds activated carbon decolorizing and filters, and after being concentrated into a small amount of crystal and separating out, adds the ethanol of 3 times of volumes of concentrated solution in the concentrated solution, and crystallisation by cooling after the centrifugal removal supernatant liquor, cleans the plane of crystal debris with ethanol, obtains the DFAIII crude product after the drying.
The DFAIII crude product that obtains is got the concentration of 100g by weight 20% to be dissolved in the deionized water, be warming up to 80 ℃, the gac that adds crude product weight 3%, decolorization filtering after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling, after the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the DFAIII finished product after the drying.
Case study on implementation 3:
300g sucrose dry product is dissolved in the 1L deionized water, with 1mol/L salt acid for adjusting pH to 7, is warming up to 50 ℃, adding sucrose fructose-transferring enzyme is 6U/g to the amount of substrate, puts into 50 ℃ of constant temperature and transforms 8 hours; Adding inulin ftructotransferase is 2U/g to the amount of substrate again, temperature 50 C, and pH5.5 reacted 24 hours, and transformation efficiency can reach 39%.The enzyme that goes out is lived, and adds activated carbon decolorizing and filters, and after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2 times of volumes of concentrated solution in the concentrated solution, and crystallisation by cooling after the centrifugal removal supernatant liquor, cleans the plane of crystal debris with ethanol, obtains the DFAIII crude product after the drying.
The DFAIII crude product that obtains is got the concentration of 100g by weight 30% to be dissolved in the deionized water, be warming up to 80 ℃, the gac that adds crude product weight 4%, decolorization filtering after being concentrated into a small amount of crystal and separating out, adds the ethanol of 4 times of concentrated solution volumes in the concentrated solution, crystallisation by cooling, after the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain finished product after the drying.
Implementation case described herein only illustrates as the present invention's spirit is tested to do with part.The technician in field of the present invention can make various modifications or replenishes or adopt similar mode to substitute described implementation case, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (1)
1. the double enzyme coupling preparation of a difructose anhydride III is characterized in that step is:
A, sucrose is dissolved in the deionized water as substrate, is made into the sucrose reaction substrate, sucrose reaction substrate concentration is controlled at 20～300g/L, regulate pH value 5～7, be warming up to 30～55 ℃, add the sucrose fructose-transferring enzyme, add-on is 1～20U/g sucrose, isothermal reaction 4～12 hours;
B, after steps A, add again inulin ftructotransferase, add-on is 2～15U/g sucrose, regulates pH value 5～7, utilizes pair enzyme coupling catalytic reactions technology, 40～60 ℃ of insulation reaction 12～36 hours, transformation efficiency reaches more than 35%;
C, with step B gained converted product solution heating, the enzyme that goes out is lived, and adds the gac of gained system quality mark 1%～3%, decolorization filtering; After being concentrated into a small amount of crystal and separating out, add the ethanol of 2～5 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling; After the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the difructose anhydride III crude product after the drying;
D, recrystallization: the difructose anhydride III crude product that the obtains concentration by massfraction 10%～30% is dissolved in the deionized water, be warming up to 70～80 ℃, the gac that adds difructose anhydride III crude product massfraction 3%, decolorization filtering after being concentrated into a small amount of crystal and separating out, adds the ethanol of 2～5 times of volumes of concentrated solution in the concentrated solution, crystallisation by cooling, after the centrifugal removal supernatant liquor, clean the plane of crystal debris with ethanol, obtain the difructose anhydride III finished product after the drying.
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| 赵萌等.新型食品添加剂双果糖酐Ⅲ的研究进展.《食品与发酵工业》.2008,第34卷(第8期),第125页第1栏第4段. * |
| 赵萌等.阳离子交换树脂分离纯化酶法合成的双果糖酐Ⅲ.《食品与发酵工业》.2009,第35卷(第9期),第134页第2栏第2段. * |
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