CN103695501A - Method for producing lactosucrose employing levansucrase - Google Patents
Method for producing lactosucrose employing levansucrase Download PDFInfo
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- CN103695501A CN103695501A CN201310660568.1A CN201310660568A CN103695501A CN 103695501 A CN103695501 A CN 103695501A CN 201310660568 A CN201310660568 A CN 201310660568A CN 103695501 A CN103695501 A CN 103695501A
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- Prior art keywords
- levansucrase
- oligomeric lactulose
- fermentation
- lactosucrose
- lactulose
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Abstract
The invention discloses a method for producing lactosucrose employing levansucrase, and belongs to the technical field of food biology. The invention relates to bacillus methylotrophicus SK21.002 screened from soil. The bacillus methylotrophicus is taken as a fermentation strain, a fermentation medium is composed of saccharose as a carbon source, and a yeast extract and peptone as nitrogen sources and the like, and the levansucrase is produced in a fermentation manner, wherein the enzyme activity can be up to 1-50U/mL by detection after fermentation; the levansucrase is added to a saccharose-lactose solution (1:1) of which the mass concentration is 10-40%, so as to synthesize lactosucrose in a catalysis manner, wherein the conversion time is 12-48 hours, and the conversion ratio can be over 10%. The lactosucrose product produced by the method disclosed by the invention is safe and reliable, and is a functional food ingredient with market potential.
Description
Technical field
The present invention relates to a kind of methylotrophy genus bacillus that can produce oligomeric lactulose (
bacillus methylotrophicus)) SK21.002 and utilize this strain fermentation to produce levansucrase, and this enzyme is prepared to the method for oligomeric lactulose (lactosucrose) for biology, belong to technical field of food biotechnology.
Background technology
Oligomeric lactulose (lactosucrose) is a kind of of functional oligose, low sugariness, low in calories, be difficult to be digested by human body, there is the bifidus bacillus of promotion propagation, suppress the physiological functions such as pathogenic bacterium generation, anti-dental caries, adjustment intestinal function, reducing blood-fat, decreasing cholesterol, promotion mineral absorption.The external commercialization oligomeric lactulose of producing is a kind of mixture that comprises oligomeric lactulose, sucrose, lactose, glucose and other micro-oligose.In recent years, functional food becomes the focus that consumers in general pay close attention to, especially the emphasis of vast food practitioner research and development.Diabetes, obesity and cardiovascular disorder crowd's increase year by year and becoming younger, makes functional food ingredient low in calories and that have a greater functionality nutritive property become the focus of concern.
Oligomeric lactulose can be widely used in numerous food as a kind of functional food additives, has broad application prospects.
Enzyme process is synthetic is the main path of suitability for industrialized production oligomeric lactulose.Enzyme process is synthetic mainly contains two kinds of methods: the one, and the beta galactose group-transfer that utilizes galactoside transferase that lactose is decomposed to produce is to the C of glucose in sucrose
4on hydroxyl, as beta-galactosidase enzymes; The 2nd, the fructosyl that utilizes fructosyl transferase that sucrose decomposition is produced is transferred to the C of lactose reducing end under neutral
1on hydroxyl, applicable enzyme has levansucrase (levansucrase) and saccharase (β-fructofuransidase).The productive rate of synthesizing lactosucrose by enzyme method is subject to various factors, and wherein the source of enzyme directly affects the productive rate of synthesizing lactosucrose by enzyme method.
The present invention uses a strain can produce the new microbe of levansucrase, utilizes this enzyme to react the oligomeric lactulose that can obtain high conversion with sucrose-lactose (1:1), and by syrup or powder concentrated, that drying and other steps obtains oligomeric lactulose.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 levansucrase that high enzyme is lived.
ー object Shi Ti of Ling of the present invention is Gong the method for this levansucrase of ー kind and sucrose-lactose (1:1) solution reaction, to obtain the oligomeric lactulose of high conversion.
A Zai ー object of the present invention is to provide a kind of oligomeric lactulose and purifies and refining method, to obtain high density oligomeric lactulose syrup or powder.
To achieve these goals, the invention provides the methylotrophy genus bacillus that a strain derives from soil (
bacillus methylotrophicus) SK21.002, this bacterial strain can produce levansucrase, and utilizes this enzyme to generate oligomeric lactulose.
Technical scheme of the present invention: a kind of method of producing oligomeric lactulose with levansucrase, while transforming sucrose-lactose production oligomeric lactulose, to sucrose: add levansucrase in sucrose-lactose substrate solution that lactose mass ratio is 1:1 and carry out catalytic production oligomeric lactulose, conversion condition is: the mass concentration of substrate solution is 10%-40%, levansucrase addition 0.5-50U/g substrate, pH5.0-8.0, conversion reaction temperature is 30-60 ℃, conversion reaction time 12-48h, transformation efficiency can reach more than 10%, obtain the enzyme reaction solution that contains oligomeric lactulose, Jin ー step processing treatment is used for preparing oligomeric lactulose syrup or oligomeric lactulose powder.
Levansucrase used derive from methylotrophy genus bacillus (
bacillus methylotrophicus) levansucrase that produces of SK21.002 strain fermentation.
In another patent ZL 201210012005.7 of the applicant " bacterial strain of levansucrase and the method for producing Polylevulosan with this enzyme are produced in a strain ", disclose fermentation method and prepared the method for levansucrase, the bacterial strain methylotrophy genus bacillus that starting strain is product levansucrase (
bacillus methylotrophicus) SK21.002, i.e. CCTCC NO:M 2011476 bacterial strains, preparation process is:
(1) seed culture
Seed culture medium: yeast extract paste l-10g/L, peptone l-20g/L, glucose l-40g/L, pH6.0-8.0, deionized water preparation;
Seed culture condition: CCTCC NO:M 2011476 bacterial strains are cultivated 10-20h and activated this bacterial strain under 30-37 ℃, the hunting speed of 150-250rpm in seed culture medium;
(2) fermentation culture
Fermention medium: yeast extract paste l-20g/L, peptone l-20g/L, sucrose l-30g/L, pH6.0-8.0, deionized water preparation;
Fermentation condition: inoculum size 1%-10%, the 10-48h that ferments in fermention medium under 30-37 ℃, the condition of stirring velocity 100-700rpm, air flow quantity 0.1-1. 0 vvm produces levansucrase;
(3) fermentation aftertreatment
Fermented liquid is collected supernatant liquor and is crude enzyme liquid after frozen centrifugation, and enzyme work reaches l-50U/mL after testing;
Gained crude enzyme liquid Jin mono-Walk ultrafiltration and concentration, or use ammonium sulfate precipitated protein matter, centrifugal and lyophilize obtains the thick enzyme powder of levansucrase.
The further processing treatment of enzyme reaction solution that contains oligomeric lactulose for decolouring, desalination, concentrated and dry.
Described decolouring is used gac or ion exchange resin to carry out.
Described desalination is undertaken by ion exchange method or membrane filter method.
The preparation method of oligomeric lactulose syrup, step is:
(1) decolouring
In containing the enzyme reaction solution of oligomeric lactulose, add the gac of enzyme reaction solution solid quality content 0.2%-2.0%, incubation 10-60min at 50-80 ℃, then carries out diatomite filtration, gets destainer;
(2) desalination
Destainer is successively carried out to desalination by cationic exchange coloum 001*7 and anionite-exchange resin 313, get demineralised liquid;
(3) concentrated
By demineralised liquid vacuum evaporation to solid quality content, be 10%-70%, obtain oligomeric lactulose syrup.
The preparation method of oligomeric lactulose powder, step is:
(1) decolouring
With the described decolouring step of oligomeric lactulose syrup preparation;
(2) desalination
With the described decolouring step of oligomeric lactulose syrup preparation;
(3) concentrated
The concentrated solution that is 10%-70% to solid quality content by demineralised liquid vacuum evaporation;
(4) spraying is dry
Control 150-200 ℃ of inlet temperature, air outlet temperature 50-100 ℃, the spraying of oligomeric lactulose concentrated solution is dry, obtain oligomeric lactulose powder.
Beneficial effect of the present invention: the present invention relates to that a strain is screened from soil and the methylotrophy genus bacillus that comes (
bacillus methylotrophicus) SK21.002, as fermentation strain, the sucrose of take forms fermention medium as carbon source, yeast extract and peptone are nitrogenous source etc., fermentative production levansucrase, and fermentation is by detection, and fermenting enzyme work reaches l-50U/mL.This levansucrase is added in mass concentration 10%-40% sucrose-lactose solution (1:1) and catalyzes and synthesizes oligomeric lactulose, transform 12-48h, transformation efficiency reaches more than 10%.The reliable ， Shi of the oligomeric lactulose product safety ー kind that the inventive method is produced has the functional food ingredient of market potential very much.
Embodiment
Be below methylotrophy genus bacillus (
bacillus methylotrophicus) SK21.002 carries out the embodiment of fermentative production levansucrase and enzymatic conversion method production oligomeric lactulose, but the present invention is not limited to listed several examples.
Embodiment 1 microorganism enzymatic production
With SK21.002, carry out cultivation and fermentation, the fermentation condition described in by specification, by being produced to the detection of enzyme the different fermentations time, find fermentation 21h after enzyme work tend to be steady, therefore determine that fermentation time is 21h left and right.
Embodiment 2 enzymatic conversion method sucrose generate oligomeric lactulose
In mass concentration 40% sucrose-lactose solution (1:1), add levansucrase catalyzed conversion, conversion condition is: pH6.5, and levansucrase addition 0.5-50U/g substrate, conversion reaction temperature is 40 ℃, conversion reaction time 24h.
The preparation of embodiment 3 oligomeric lactulose syrup
(1) decolouring
In containing the enzyme reaction solution of oligomeric lactulose, add the gac of enzyme reaction solution solid quality content 0.2%-2.0%, incubation 10-60min at 50-80 ℃, then carries out diatomite filtration, gets destainer;
(2) desalination
Destainer is successively carried out to desalination by cationic exchange coloum 001*7 and anionite-exchange resin 313, get demineralised liquid;
(3) concentrated
By demineralised liquid vacuum evaporation to solid quality content, be 10%-70%, obtain oligomeric lactulose syrup.
The preparation of embodiment 4 oligomeric lactulose powder
(1) decolouring
Decolorization condition with embodiment 3;
(2) desalination
Desalination condition with embodiment 3;
(3) concentrated
The concentrated solution that is 10%-70% to solid quality content by demineralised liquid vacuum evaporation;
(4) spraying is dry
Control 150-200 ℃ of inlet temperature, air outlet temperature 50-100 ℃, the spraying of oligomeric lactulose concentrated solution is dry, obtain oligomeric lactulose powder.
Claims (4)
1. a method of producing oligomeric lactulose with levansucrase, it is characterized in that to sucrose: in sucrose-lactose substrate solution that lactose mass ratio is 1:1, add levansucrase catalytic production oligomeric lactulose, conversion condition is: the solute mass concentration of substrate solution is 10%-40%, levansucrase addition 0.5-50U/g substrate, pH 5.0-8.0, conversion reaction temperature is 30-60 ℃, conversion reaction time 12-48h, obtain the enzyme reaction solution that contains oligomeric lactulose, further processing treatment is used for preparing oligomeric lactulose syrup or oligomeric lactulose powder,
Levansucrase used derive from methylotrophy genus bacillus (
bacillus methylotrophicus) levansucrase that produces of SK21.002 strain fermentation.
2. method according to claim 1, it is characterized in that the further processing treatment of the enzyme reaction solution that contains oligomeric lactulose for decolouring, desalination, concentrated and dry.
3. method according to claim 2, is characterized in that described decolouring use gac or ion exchange resin carry out.
4. method according to claim 2, is characterized in that described desalination undertaken by ion exchange method or membrane filter method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201310660568.1A CN103695501A (en) | 2013-12-10 | 2013-12-10 | Method for producing lactosucrose employing levansucrase |
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| CN201310660568.1A CN103695501A (en) | 2013-12-10 | 2013-12-10 | Method for producing lactosucrose employing levansucrase |
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Cited By (5)
| 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 |
| CN104805152A (en) * | 2014-12-04 | 2015-07-29 | 山东百龙创园生物科技有限公司 | Enzyme method for production of lactulose |
| CN106191157A (en) * | 2016-08-29 | 2016-12-07 | 山东百龙创园生物科技有限公司 | A kind of preparation method of high-purity oligomeric lactulose |
| CN106434504A (en) * | 2016-09-22 | 2017-02-22 | 江南大学 | Recombinant gene engineering strain for expressing levansucrase and application thereof |
| TWI624547B (en) * | 2017-01-26 | 2018-05-21 | 國立中興大學 | Preparation method of lactosucrose |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0447125A1 (en) * | 1990-03-08 | 1991-09-18 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Process for preparing lactosucrose high-content powder and use of said powder |
| CN101775423A (en) * | 2010-02-08 | 2010-07-14 | 南昌大学 | Method for synthesizing lactosucrose by enzyme method |
| CN102533605A (en) * | 2012-01-16 | 2012-07-04 | 江南大学 | Strain capable of producing levansucrase and method for producing levan by using levansucrase |
-
2013
- 2013-12-10 CN CN201310660568.1A patent/CN103695501A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0447125A1 (en) * | 1990-03-08 | 1991-09-18 | Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo | Process for preparing lactosucrose high-content powder and use of said powder |
| CN101775423A (en) * | 2010-02-08 | 2010-07-14 | 南昌大学 | Method for synthesizing lactosucrose by enzyme method |
| CN102533605A (en) * | 2012-01-16 | 2012-07-04 | 江南大学 | Strain capable of producing levansucrase and method for producing levan by using levansucrase |
Cited By (8)
| 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 |
| CN104805152A (en) * | 2014-12-04 | 2015-07-29 | 山东百龙创园生物科技有限公司 | Enzyme method for production of lactulose |
| CN106191157A (en) * | 2016-08-29 | 2016-12-07 | 山东百龙创园生物科技有限公司 | A kind of preparation method of high-purity oligomeric lactulose |
| CN106191157B (en) * | 2016-08-29 | 2019-07-09 | 山东百龙创园生物科技股份有限公司 | A kind of preparation method of lactosucrose |
| CN106434504A (en) * | 2016-09-22 | 2017-02-22 | 江南大学 | Recombinant gene engineering strain for expressing levansucrase and application thereof |
| CN106434504B (en) * | 2016-09-22 | 2019-06-07 | 江南大学 | It is a kind of recombinantly express levansucrase engineering strain and its application |
| TWI624547B (en) * | 2017-01-26 | 2018-05-21 | 國立中興大學 | Preparation method of lactosucrose |
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Application publication date: 20140402 |