CN104087537A - Strain for producing inulin fructose transferase and method for producing difructose anhydride III by using transferase - Google Patents
Strain for producing inulin fructose transferase and method for producing difructose anhydride III by using transferase Download PDFInfo
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- CN104087537A CN104087537A CN201410327029.0A CN201410327029A CN104087537A CN 104087537 A CN104087537 A CN 104087537A CN 201410327029 A CN201410327029 A CN 201410327029A CN 104087537 A CN104087537 A CN 104087537A
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- difructose anhydride
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Abstract
The invention relates to a strain for producing an inulin fructosyltransferase and a method for producing a difructose anhydride III by using the transferase, belonging to the biotechnology field of foods. The invention relates to a strain for producing inulin fructosyltransferase; a classification name of the strain is arthrobacter chlorophenolicus SK 33.001; the strain is preserved in Typical Culture Preservation Centre in China; the preservation number is CCTCC NO: M 2013387. The strain is taken as a starting strain and used for composing a fermentation culture medium with a nitrogen source and inorganic salt by taking insulin as a carbon source; the fermentation culture medium is used for producing the inulin fructosyltransferase by fermentation. The fermentation culture medium takes the insulin and sodium nitrate as the main carbon source and the nitrogen source; the enzyme activity of the inulin fructosyltransferase in fermentation broth can achieve 2 to 100U/ml through the detection after the fermentation. When the inulin fructosyltransferase is added to a 1%-30% insulin solution and used for producing the difructose anhydride III, the conversion is carried out for 3 to 24h, and the conversation rate reaches over 75%. The obtained difructose anhydride III product is safe and reliable, thereby being a functional sweetener with great market potentiality.
Description
Technical field
The present invention relates to produce a kind of new microorganism and the cultivation and fermentation production inulin ftructotransferase thereof of inulin ftructotransferase, and this enzyme is used for transforming the method that synanthrin is prepared difructose anhydride III, belong to technical field of food biotechnology.More particularly, the present invention relates to a strain laboratory preservation chlorophenol Arthrobacter (
arthrobacter chlorophenolicus) SK 33. 001, it can produce inulin ftructotransferase, and the difructose anhydride III that utilizes this inulin ftructotransferase to transform synanthrin to generate following structure (α-D-fructofuranose-2 ', 1:2,3 '-β-D-fructofuranose dianhydride).
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.And diabetes, obesity and cardiovascular disorder crowd's increase year by year and becoming younger makes low in caloriesly, the functional sweetener with greater functionality nutritive property becomes the focus of concern.
Difructose anhydride III (Difructose anhydride) is a kind of novel irreducibility disaccharide, is present on a small quantity in the plants such as witloof, jerusalem artichoke at occurring in nature, comes across on a small quantity in the course of processing of honey, coffee etc.Its relative sweetness is 52% of sucrose, and caloric value only has 1/15 (0.263 kcal/g) of sucrose; Difructose anhydride III is very stable to heat and acid, under normal food processing conditions, there will be hardly brown stain or decomposing phenomenon, the high temperature infusion in ability hard candy when production and not brown stain.Simultaneously difructose anhydride III also has good functional performance, as do not absorbed at digestive tube, and generate energy not, can be used as a kind of sweeting agent and be used as the assisting therapy of losing weight; As a kind of increasing discrimination factor, can promote the growth of enteric microorganism, obviously improve defecation, diuresis function; As the functional sugar that promotes that mineral element absorbs, can make the mineral elements such as Ca, Mg, Zn, the Cu degree of utilizing that is absorbed by the body greatly improve, promote bone growth.These advantages hold out broad prospects its application in foodstuffs industry.
Because difructose anhydride III is extremely low at natural content, natural extract separation costs is high, be not suitable for the requirement of industrialized production, chemical catalysis has the disadvantageous factors such as many by products of formation and chemical pollutant, make cheaply, the conversion technology that transformation efficiency is high becomes the study hotspot of producing difructose anhydride III.
From Uchiyama in 1973, found a kind of novel inulinase (inulase П) first from produce urea Arthrobacter, the research of using biotransformation method to prepare difructose anhydride III has had the history of more than 30 years.Up to now, found that more than ten plant microorganism and can produce this enzyme, mainly concentrated on genus arthrobacter, comprised
arthrobacter ureafaciens7116,
arthrobacter ilicisoKU17B,
arthrobacter globiformisc11-1,
arthrobactersp
.h65-7,
arthrobactersp
.a-6,
arthrobacter pascenst13-2,
arthrobactersp. Bu0141,
arthrobactersp. L68-1 has also found this enzyme in addition in three kinds of other Pseudomonas, as
flavobacteriumsp. LC-413,
bacillussp. snu-7,
leifsoniasp. T88-4.
The inventor has investigated and has studied prior art further and the method for various high produced in yields difructose anhydride IIIs has been studied, finally we screen the new microbe that a strain can produce inulin ftructotransferase, proved by this enzyme and reacted the difructose anhydride III that can access high conversion with the synanthrin of high density, and made the difructose anhydride III of syrup, powder or three kinds of forms of crystal by concentrated, the method dry, crystallization of spraying.Based on above-mentioned discovery, the present invention has been proposed.
Summary of the invention
The object of this invention is to provide a kind of new microorganism, it can produce the inulin ftructotransferase that high enzyme is lived.
Another object of the present invention is to provide a kind of method of this inulin ftructotransferase and high density synanthrin solution reaction, to obtain the difructose anhydride III of high conversion.
A further object of the present invention is to provide a kind of difructose anhydride III and purifies and refining method, to obtain difructose anhydride III syrup, powder or crystal.
To achieve these goals, the invention provides a kind of chlorophenol Arthrobacter by laboratory preservation (
arthrobacter chlorophenolicus) SK 33. 001, can produce the bacterial strain of inulin ftructotransferase, and utilize this inulin ftructotransferase to transform synanthrin generation difructose anhydride III.The present invention adopts
arthrobacter chlorophenolicusthe inulin ftructotransferase of SK 33. 001 fermentative production, enzyme is lived higher, synanthrin can be converted into difructose anhydride III, and difructose anhydride III transformation efficiency is up to more than 75%, by product is few, except difructose anhydride III and unconverted synanthrin, only has a small amount of oligofructose.
Technical scheme of the present invention: the bacterial strain of inulin ftructotransferase is produced in a strain, its Classification And Nomenclature be chlorophenol Arthrobacter (
arthrobacter chlorophenolicus) SK 33. 001, being preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2013387.
With described microorganism chlorophenol Arthrobacter SK 33. 001, the method for fermentative production inulin ftructotransferase, step is:
(1) seed culture
Seed culture medium: peptone 1-10g/L, sodium-chlor 1-10g/L, extractum carnis 1-5g/L, pH 7.0, deionized water preparation;
Seed culture condition: by chlorophenol Arthrobacter SK 33. 001 access seed culture mediums, cultivate 12-24 h under 25-30 ℃, the oscillating condition of 160-240 rpm;
(2) fermentation culture
Fermention medium: synanthrin 5-50g/L, SODIUMNITRATE 1-20g/L, yeast extract paste 1-5g/L, dipotassium hydrogen phosphate 0.5-2g/L, magnesium sulfate 0.5-3g/L, iron vitriol 0.01-0.05g/L, pH 6.5-8.0, deionized water preparation;
Fermentation condition: seed liquor inoculum size is 1%-5% is that 200-500rpm, air flow quantity are 5-30m at 25-30 ℃, stirring velocity
3the 12-84h that ferments in fermention medium under/condition (Lh) produces inulin ftructotransferase;
(3) fermentation aftertreatment
By centrifuging, remove the thalline in fermented liquid, obtain inulin ftructotransferase crude enzyme liquid; Or the further ultrafiltration and concentration of inulin ftructotransferase crude enzyme liquid, with the industrial spirit precipitating proteins of 3 times of concentrated solution volumes, centrifugal and lyophilize obtains the thick enzyme powder of inulin ftructotransferase.
With the inulin ftructotransferase of preparation, transform the method that synanthrin is produced difructose anhydride III, in the synanthrin solution of mass concentration 1%-30%, add inulin ftructotransferase catalyzed conversion, conversion reaction conditions is: substrate synanthrin concentration range is 1%-30%, enzyme is 0.01-10 U/g to the consumption of substrate, pH4.0-8.0, conversion reaction temperature is 30-50 ℃, conversion reaction time 3-24h, obtain the enzyme reaction solution that contains difructose anhydride III, for the preparation of difructose anhydride III syrup, difructose anhydride III powder or difructose anhydride III crystal.
The preparation method of difructose anhydride III syrup, step is:
(1) decolouring
In containing the enzyme reaction solution of difructose anhydride III, add the gac of enzyme reaction solution solid content 0.2%-2.0%, incubation 30min at 80 ℃, then carries out diatomite filtration, gets destainer;
(2) concentrated
By destainer vacuum evaporation to solid content, be that 60%-95% obtains difructose anhydride III syrup.
The preparation method of difructose anhydride III powder, step is:
(1) decolouring
With preparing the step of decolouring described in difructose anhydride III syrup;
(2) concentrated
The concentrated solution that is 20%-40% to solid content by destainer vacuum evaporation;
(3) spraying thousand is dry
Maltodextrin is mixed according to the ratio of weight ratio 1:1-5 with the difructose anhydride III concentrated solution in solid content butt, and 170-180 ℃ of inlet temperature of control, air outlet temperature are 70-80 ℃, and spraying is dry, obtains the difructose anhydride III powder containing maltodextrin.
The preparation method of difructose anhydride III crystal, step is:
(1) decolouring
With preparing the step of decolouring described in difructose anhydride III syrup;
(2) crystallization
It is 60%-95% that destainer is concentrated into solid content, fast cooling to 50 ℃, slowly evenly add difructose anhydride III crystal seed, to the ethanol that slowly adds 0.2-2 times of concentrated solution volume in concentrated solution, by the slow cooling process temperature of 12h, be down to 10 ℃, obtain the massecuite of difructose anhydride-containing III crystal, with ethanol, clean the impurity of removing plane of crystal, centrifugation, vacuum-drying, to constant weight, obtains difructose anhydride III crystal.
Detect the method for difructose anhydride III content:
Get enzyme reaction solution centrifugal, supernatant liquor filtering with microporous membrane (0.22 μ m), on filtrate, HPLC analyzes.HPLC condition: Sugarpakl, 6.5 mmid * 300 mm calcium type cationic exchange coloums, pure water is made moving phase, differential refraction detector, 85 ℃ of column temperatures, flow velocity: 0.4 ml/min, sample size: 10 μ L, difructose anhydride III standard specimen concentration: 0.5%.
Beneficial effect of the present invention: the present invention screens the new microbe CCTCC NO:M 2013387 that a strain can produce inulin ftructotransferase, has proved by inulin ftructotransferase and has reacted the difructose anhydride III that can access high conversion with the synanthrin of high density; And obtained the difructose anhydride III of syrup, powder or three kinds of forms of crystal by concentrated, the method dry, crystallization of spraying.
biological material specimens preservation
The bacterial strain of inulin ftructotransferase is produced in one strain, its Classification And Nomenclature be chlorophenol Arthrobacter (
arthrobacter chlorophenolicus) SK 33. 001, being preserved in Chinese Typical Representative culture collection center, cylinder claims CCTCC, address: Wuhan, China Wuhan University, its deposit number is CCTCC NO:M 2013387, preservation date is on September 2nd, 2013.
Embodiment
Be below the embodiment that chlorophenol Arthrobacter SK 33. 001 carries out fermentative production inulin ftructotransferase and enzymatic conversion method production difructose anhydride III, but the present invention is not limited to listed several examples.
Embodiment 1 fermentation time is on producing the impact of inulin ftructotransferase
Fermentation condition described in by specification, by right
arthrobacter chlorophenolicussK 33. 001 produces the detection of IFTase in the different fermentations time, find that fermentation 24h IFTase enzyme is alive the highest, and along with fermentation time extends, IFTase enzyme is lived and slightly declined, therefore determine that fermentation time is about 24h.Fermentation time is as shown in table 1 on the impact of product inulin ftructotransferase.Enzyme activity is defined as at temperature 70 C, and it is 1 enzyme activity unit that pH6.0 condition produces the required enzyme amount of 1.0 μ mol difructose anhydride III (DFA Ш) in lower 1 minute.
Table 1 fermentation time is produced the impact of difructose anhydride III on enzymatic conversion method
Embodiment 2 synanthrin concentration are produced the impact of difructose anhydride III on enzymatic conversion method
Enzymatic conversion method condition described in by specification, synanthrin is mixed with to the aqueous solution of different concns, under 70 ℃, pH 6.0 conditions, reacts 24h, found that the increase along with synanthrin concentration, transformation efficiency constantly declines, but transformation efficiency still can guarantee more than 75% under 30% synanthrin concentration of aqueous solution.The impact that synanthrin concentration is produced difructose anhydride III to enzymatic conversion method is as shown in table 2.
Table 2 synanthrin concentration is produced the impact of difructose anhydride III on enzymatic conversion method
The preparation of embodiment 3 difructose anhydride III syrup
(1) decolouring
To in the enzyme reaction solution that contain difructose anhydride III, add the gac of enzyme reaction solution solid content 0.2%-2%, incubation 30min at 80 ℃, then carries out diatomite filtration, gets destainer;
(2) concentrated
By destainer vacuum evaporation to solid content, be 60%-95%, obtain difructose anhydride III syrup.
The preparation of embodiment 4 difructose anhydride III powder
(1) decolouring
To in the enzyme reaction solution that contain difructose anhydride III, add the gac of enzyme reaction solution solid content 0.2%-2.0%, incubation 30min at 80 ℃, then carries out diatomite filtration, gets destainer;
(2) concentrated
The concentrated solution that is 20%-40% to solid content by destainer vacuum evaporation;
(3) spraying is dry
Maltodextrin is mixed according to the ratio of weight ratio 1:1-5 with the difructose anhydride III concentrated solution in solid content butt, and 170-180 ℃ of inlet temperature of control, air outlet temperature are 70-80 ℃, and spraying is dry, obtains the difructose anhydride III powder containing maltodextrin.
The impact of embodiment 5 ethanol additions on the nucleus form of difructose anhydride III crystal
To the phenomenon of graining process of different ethanol additions and the description of nucleus in Table 3, in the suspension of the difructose anhydride III nucleus of preparing, choose appropriate sample liquid from graining, with through the saturated glycerine of difructose anhydride III, sample liquid being diluted to proper concn, the diluted sample that takes a morsel again liquid, examines under a microscope nucleus shape
State.
The impact of table 3 ethanol addition on difructose anhydride III nucleus form
。
Claims (6)
1. the bacterial strain of inulin ftructotransferase is produced in a strain, its Classification And Nomenclature be chlorophenol Arthrobacter (
arthrobacter chlorophenolicus) SK 33. 001, being preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:M 2013387.
2. with microorganism chlorophenol Arthrobacter SK 33. 001 claimed in claim 1, the method for fermentative production inulin ftructotransferase, the steps include:
(1) seed culture
Seed culture medium: peptone 1-10g/L, sodium-chlor 1-10g/L, extractum carnis 1-5g/L, pH 7.0, deionized water preparation;
Seed culture condition: by chlorophenol Arthrobacter SK 33.001 access seed culture mediums, cultivate 12-24h under 25-30 ℃, the oscillating condition of 160-240 rpm;
(2) fermentation culture
Fermention medium: synanthrin 5-50g/L, SODIUMNITRATE 1-20g/L, yeast extract paste 1-5g/L, dipotassium hydrogen phosphate 0.5-2g/L, magnesium sulfate 0.5-3g/L, iron vitriol 0.01-0.05g/L, pH 6.5-8.0, deionized water preparation;
Fermentation condition: seed liquor inoculum size is 1%-5% is that 200-500rpm, air flow quantity are 5-30m at 25-30 ℃, stirring velocity
3the 12-84h that ferments in fermention medium under/condition (Lh) produces inulin ftructotransferase;
(3) fermentation aftertreatment
By centrifuging, remove the thalline in fermented liquid, obtain inulin ftructotransferase crude enzyme liquid; By the further ultrafiltration and concentration of inulin ftructotransferase crude enzyme liquid, with the industrial spirit precipitating proteins of 3 times of concentrated solution volumes, centrifugal and lyophilize obtains the thick enzyme powder of inulin ftructotransferase.
3. the inulin ftructotransferase of preparing by method described in claim 2 transforms the method that synanthrin is produced difructose anhydride III, it is characterized in that:
In mass concentration 1%-30% synanthrin solution, add inulin ftructotransferase catalyzed conversion, conversion reaction conditions is: substrate synanthrin concentration range is 1%-30%, enzyme is 0.01-10U/g to the consumption of substrate, pH 4.0-8.0, conversion reaction temperature is 30-50 ℃, conversion reaction time 3-24h, obtains the enzyme reaction solution that contains difructose anhydride III, for the preparation of difructose anhydride III syrup, difructose anhydride III powder or difructose anhydride III crystal.
4. method according to claim 3, is characterized in that preparing difructose anhydride III syrup, and step is:
(1) decolouring
In containing the enzyme reaction solution of difructose anhydride III, add the gac of enzyme reaction solution solid content 0.2%-2%, incubation 30min at 80 ℃, then carries out diatomite filtration, gets destainer;
(2) concentrated
By destainer vacuum evaporation to solid content, be 60%-95%, obtain difructose anhydride III syrup.
5. method according to claim 3, is characterized in that preparing difructose anhydride III powder, and step is:
(1) decolouring
With the step of decolouring described in claim 4;
(2) concentrated
The concentrated solution that is 20%-40% to solid content by destainer vacuum evaporation;
(3) spraying is dry
Maltodextrin is mixed according to the ratio of weight ratio 1 ︰ 1-5 with the difructose anhydride III concentrated solution in solid content butt, 170-180 ℃ of inlet temperature of control, air outlet temperature are 70-80 ℃, spraying is dry, obtains the difructose anhydride III powder containing maltodextrin.
6. method according to claim 3, is characterized in that preparing difructose anhydride III crystal, and step is:
(1) decolouring
With the step of decolouring described in claim 4;
(2) crystallization
It is 60%-95% that destainer is concentrated into solid content, fast cooling to 50 ℃, slowly evenly add difructose anhydride III crystal seed, to the ethanol that slowly adds 0.2-2 times of concentrated solution volume in concentrated solution, by the slow cooling process temperature of 12 h, be down to 10 ℃, obtain the massecuite of difructose anhydride-containing III crystal, with ethanol, clean the impurity of removing plane of crystal, centrifugation, vacuum-drying, to constant weight, obtains difructose anhydride III crystal.
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| CN201410327029.0A CN104087537B (en) | 2014-07-10 | 2014-07-10 | One strain is produced the bacterial strain of inulin ftructotransferase and is produced the method for difructose anhydride III with this enzyme |
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Cited By (3)
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| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN105255772A (en) * | 2015-11-06 | 2016-01-20 | 江南大学 | Strain for producing inulin fructotransferase, method for producing inulin fructotransferase by virtue of strain and application of strain |
| CN105506034A (en) * | 2016-01-26 | 2016-04-20 | 江南大学 | Method for efficient synthesis of difructose anhydride III |
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2014
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073456A (en) * | 2014-07-10 | 2014-10-01 | 江南大学 | Bacterial strain for producing levansucrase and method for producing lactosucrose by utilizing lavansucrase |
| CN104073456B (en) * | 2014-07-10 | 2016-04-13 | 江南大学 | One strain is produced the bacterial strain of levansucrase and is produced the method for oligomeric lactulose with this enzyme |
| CN105255772A (en) * | 2015-11-06 | 2016-01-20 | 江南大学 | Strain for producing inulin fructotransferase, method for producing inulin fructotransferase by virtue of strain and application of strain |
| CN105255772B (en) * | 2015-11-06 | 2019-09-13 | 江南大学 | One plant produces the bacterial strain of inulin ftructotransferase and its produces the methods and applications of inulin ftructotransferase |
| CN105506034A (en) * | 2016-01-26 | 2016-04-20 | 江南大学 | Method for efficient synthesis of difructose anhydride III |
| WO2017128529A1 (en) * | 2016-01-26 | 2017-08-03 | 江南大学 | Highly efficient method for synthesizing difructose anhydride iii |
| CN105506034B (en) * | 2016-01-26 | 2019-01-11 | 江南大学 | A method of synthesis difructose anhydride III |
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