CN108866125A - A method of reducing trehalose turbidity - Google Patents
A method of reducing trehalose turbidity Download PDFInfo
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- CN108866125A CN108866125A CN201810970926.1A CN201810970926A CN108866125A CN 108866125 A CN108866125 A CN 108866125A CN 201810970926 A CN201810970926 A CN 201810970926A CN 108866125 A CN108866125 A CN 108866125A
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- C12P19/00—Preparation of compounds containing saccharide radicals
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Abstract
The invention discloses a kind of methods for reducing trehalose turbidity, belong to separation and extraction technology field.Method of the invention as will using trehalase reaction solution made from trehalose synthase conversion method successively through carbohydrase enzymatic hydrolysis, the filtering of first time active carbon, weak anion resin filtering, acidulous cation resin filtering, second of active carbon filtering, alcohol filter and etc. processing, the purity of trehalose finished product is promoted to 99.8% from 78.1% by success, and turbidity is reduced to 0.001 from 0.06.
Description
Technical field
The present invention relates to a kind of methods for reducing trehalose turbidity, belong to separation and extraction technology field.
Background technique
Trehalose is irreducibility disaccharide made of being closed by two glucose molecules by the bond of α, α -1,1, is widely present
In bacterium, fungi, algae, rudimentary plant and insect.It has been investigated that the sugar has unique biological function, there is protection
Large biological molecule, protect cell membrane, protected protein matter from freezing, drying and osmotic pressure variation etc. caused by destruction effect,
Extensive use is all had in fields such as food, medicine, cosmetics, agriculturals.
Currently, demand of the domestic and international market to trehalose constantly expands, production technology is also constantly being followed after progress.It is early
Phase, seaweed sugar product from yeast cells mainly by extracting, and low yield is at high cost, complex process, limits answering for trehalose
With；In recent years, the trehalose synthase that a step converts maltose generation trehalose is capable of in discovery in many microorganisms, and the approach is not
Anakinetomer is consumed, does not depend on phosphoric acid, and substrate maltose price is low, it is readily available, there is biggish price compared with trehalose
Space.Therefore, which has industrial prospect.
As Chinese patent literature CN101831474A (application number 201010137964.2) discloses a kind of utilization stench vacation
The method that monad produces trehalose, including pseudomonas putida incubation step, beta -mercaptoethanol processing step and fermentation step.
The invention is using maltose as substrate, will using its intracellular trehalose synthase by preparing the permeability cell of pseudomonas putida
One step of maltose is converted into trehalose.After measured, with beta -mercaptoethanol handle pseudomonas putida permeability cell solution in about
There is 3%~5% trehalose to generate.
Currently, also containing maltose, Portugal other than trehalose in enzyme transforming process production trehalose enzyme reaction solution obtained
The carbohydrates such as grape sugar, in addition there are the impurity such as albumen, pigment and metal ion.Therefore it needs just obtain by serial purification process
The higher trehalose of purity.
But due to complicated component sugared in the reaction solution in enzyme transforming process, separation means in the prior art are not
Reaction solution suitable for enzyme transforming process.
Summary of the invention
To solve the above problems, the present invention provides a kind of methods for reducing trehalose turbidity.The method will be by that will utilize
Trehalase reaction solution made from trehalose synthase conversion method is successively through carbohydrase enzymatic hydrolysis, the filtering of first time active carbon, alkalescent
Resin anion (R.A.) filtering, acidulous cation resin filtering, the filtering of second active carbon, alcohol filter and etc. processing, successfully will be extra large
The purity of algae sugar finished product is promoted to 99.8% from 78.1%, and turbidity is reduced to 0.001 from 0.06.
Technical scheme is as follows：
The present invention provides a kind of methods for reducing trehalose turbidity, and the method comprises the following steps：
(1) 80~100 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, heat preservation 10~
30 minutes, then carbohydrase was added in cooling, carbohydrase additional amount is 12~24U/ml, adjusted pH 4.0~7.0, control saccharification temperature
Degree is 60~65 DEG C, 8~12h of enzymolysis time, then heats to 80~100 DEG C, keeps the temperature 10~30 minutes, and seaweed is made in filtering
Sugared crude liquid；
(2) quality is added into trehalose crude liquid made from step (1) account for trehalose crude liquid gross mass 0.5%~3%
Active carbon decolourizes 20~60 minutes, and filtering obtains first-time filtrate；
(3) step (2) it is obtained obtain adding quality in first-time filtrate account for first-time filtrate gross mass 0.5~0.8%
Citric acid, 0.1~0.5% NaCl and 6~8% ZnCl2Afterwards successively by weak anion resin and faintly acid sun
It is filtered after ion exchange resin, obtains secondary filtrate；
(4) activity that quality accounts for secondary filtrate gross mass 0.5%~3% is added in the secondary filtrate made from step (3)
Charcoal filters after being decolourized, and decolourizes 20~60 minutes, and filtering obtains filtrate three times；
(5) quality is added in step (4) filtrate three times obtained and accounts for filtrate gross mass 0.3~0.8% three times
Na2CO3, 8~12% ethyl alcohol after stand 0.5~1.5 hour, filter, obtain four filtrates；
(6) the anhydrous second that quality accounts for four filtrate gross masses 5~10% is added in four filtrates made from step (5)
Alcohol, 0.1~3% crystal seed and be cooled to 20~30 DEG C of progress spontaneous nucleations after carrying out concentration graining, after centrifugation wet crystallization；
(7) the wet crystallization that step (6) obtains is dried, obtains trehalose finished product.
In one embodiment of the invention, the method comprises the following steps：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%
Acid, 0.3% NaCl and 6.5% ZnCl2Afterwards successively after weak anion resin and acidulous cation resin
It is filtered, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.6% three times is added in step (4) filtrate three times obtained2CO3, 10%
Ethyl alcohol after stand 1 hour, filter, obtain four filtrates；
(6) quality is added in four filtrates made from step (5) and accounts for the dehydrated alcohol of four filtrate gross masses 8%, 2%
Crystal seed and be cooled to 25 DEG C of progress spontaneous nucleations after carrying out concentration graining, after centrifugation wet crystallization；
(7) the wet crystallization that step (6) obtains is dried, obtains trehalose finished product.
In one embodiment of the invention, in the step (3), first-time filtrate is followed by weakly-basic anion tree
The speed of rouge and acidulous cation resin is 15~20mL/min.
In one embodiment of the invention, in the step (3), first-time filtrate is followed by weakly-basic anion tree
The speed of rouge and acidulous cation resin is 15mL/min.
In one embodiment of the invention, the concentration of ethyl alcohol is 70% in the step (5).
In one embodiment of the invention, in the step (6), the temperature of concentration is 45~55 DEG C.
In one embodiment of the invention, in the step (6), the temperature of concentration is 50 DEG C.
In one embodiment of the invention, in the step (6), the time of crystallization is 60~70h.
In one embodiment of the invention, in the step (6), crystallization time 60h.
In one embodiment of the invention, in the step (6), the time of centrifugation is 10~20min.
In one embodiment of the invention, in the step (6), the time of centrifugation is 16min.
In one embodiment of the invention, in the step (6), 2500~3500r/min of revolving speed of centrifugation.
In one embodiment of the invention, in the step (6), the revolving speed 2800r/min of centrifugation.
In one embodiment of the invention, the drying in the step (7) is by wet crystallization in 75~85 DEG C of baking
Dry 6~10h in case.
In one embodiment of the invention, the drying in the step (7) is to crystallize wet in 80 DEG C of baking oven
Dry 8h.
The present invention provides a kind of above-mentioned application of the method in terms of preparing trehalose for reducing trehalose turbidity.
Beneficial effect：
Method of the invention will be as that will utilize trehalase reaction solution made from trehalose synthase conversion method successively through being saccharified
Enzyme enzymatic hydrolysis, the filtering of first time active carbon, weak anion resin filtering, acidulous cation resin filtering, second of activity
Charcoal filtering, alcohol filter and etc. processing, the purity of trehalose finished product is successfully promoted to 99.8% from 78.1%, turbidity is from 0.06 drop
Down to 0.001.
Specific embodiment
The present invention will be further elaborated with comparative example combined with specific embodiments below.
Trehalase reaction solution involved in following embodiments be recorded in Publication No. CN107227304A, application No. is
Escherichia coli recombinant strain of the utilization containing mutant of trehalose synthetase M106K in 201710585267.5 patent document passes through
(for details, reference can be made to the embodiments 1 of this patent document) obtained after fermentation.
Detection method involved in following embodiments is as follows：
Trehalose method for detecting purity：High performance liquid chromatography.
Trehalose turbidity detection method：Referring to GB/T 23529-2009 trehalose.
Embodiment 1
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%
Acid, 0.3% NaCl and 6.5% ZnCl2301 resin anion (R.A.)s and 116 sun are passed through successively with the flow velocity of 15mL/min afterwards
It is filtered after ion exchange resin, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.6% three times is added in step (4) filtrate three times obtained2CO3, 10%
70% ethyl alcohol after stand 1 hour, filter, obtain four filtrates；
(6) 8% dehydrated alcohol, 2% crystal seed are added in four filtrates made from step (5), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 99.8%, turbidity 0.001.
Embodiment 2
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) activity that quality accounts for trehalose crude liquid gross mass 0.5% is added into trehalose crude liquid made from step (1)
Charcoal decolourizes 45 minutes, and filtering obtains first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.5%
Acid, 0.1% NaCl and 6% ZnCl2Afterwards with the flow velocity of 15mL/min successively pass through 301 resin anion (R.A.)s and 116 sun from
It is filtered after subtree rouge, obtains secondary filtrate；
(4) the active carbon progress that quality accounts for secondary filtrate gross mass 0.5% is added in the secondary filtrate made from step (3)
It filters, decolourizes 45 minutes after decoloration, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.3% three times is added in step (4) filtrate three times obtained2CO3, 8%
Ethyl alcohol after stand 0.5 hour, filter, obtain four filtrates；
(6) 5% dehydrated alcohol, 0.1% crystal seed are added in four filtrates made from step (5), is carried out in 50 DEG C
25 DEG C of progress spontaneous nucleations are cooled to after concentration graining, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet knot
It is brilliant；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 99.2%, turbidity 0.003.
Embodiment 3
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 3% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.8%
Acid, 0.5% NaCl and 8% ZnCl2Afterwards with the flow velocity of 15mL/min successively pass through 301 resin anion (R.A.)s and 116 sun from
It is filtered after subtree rouge, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 3% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.8% three times is added in step (4) filtrate three times obtained2CO3, 12%
70% ethyl alcohol after stand 1 hour, filter, obtain four filtrates；
(6) 10% dehydrated alcohol, 3% crystal seed are added in four filtrates made from step (5), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 99.8%, turbidity 0.001.
Embodiment 4
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%
Acid, 0.3% NaCl and 6.5% ZnCl2301 resin anion (R.A.)s and 116 sun are passed through successively with the flow velocity of 20mL/min afterwards
It is filtered after ion exchange resin, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.6% three times is added in step (4) filtrate three times obtained2CO3, 10%
70% ethyl alcohol after stand 1 hour, filter, obtain four filtrates；
(6) 8% dehydrated alcohol, 2% crystal seed are added in four filtrates made from step (5), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 99.6%, turbidity 0.002.
Comparative example 1
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) crystal seed obtained for obtaining first-time filtrate and being added 2%, cool down after carrying out concentration graining in 50 DEG C
To 25 DEG C of progress spontaneous nucleations, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(4) the wet crystallization for obtaining step (3) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 78.1%, turbidity 0.2.
Comparative example 2
Specific step is as follows：
(2) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) activity that quality accounts for trehalose crude liquid gross mass 0.5% is added into trehalose crude liquid made from step (1)
Charcoal decolourizes 45 minutes, and filtering obtains first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.5%
Acid, 0.1% NaCl and 6% ZnCl2It is successively laggard by weak anion resin and acidulous cation resin afterwards
Row filtering, obtains secondary filtrate；
(4) the active carbon progress that quality accounts for secondary filtrate gross mass 0.5% is added in the secondary filtrate made from step (3)
It filters, decolourizes 45 minutes after decoloration, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.3% three times is added in step (4) filtrate three times obtained2CO3, 8%
Ethyl alcohol after stand 0.5 hour, filter, obtain four filtrates；
(6) 5% dehydrated alcohol, 0.1% crystal seed are added in four filtrates made from step (5) and be concentrated
It is cooled to 25 DEG C of progress spontaneous nucleations after crystalline substance, wet crystallization is obtained after centrifugation；
(7) the wet crystallization that step (6) obtains is dried, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 89.3%, turbidity 0.09.
Comparative example 3
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) Na obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%2CO3、
1 hour is stood after 10% 70% ethyl alcohol, filters, obtains secondary filtrate；
(4) 8% dehydrated alcohol, 2% crystal seed are added in four filtrates made from step (3), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(5) the wet crystallization for obtaining step (4) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 87.3%, turbidity 0.1.
Comparative example 4
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%
Acid, 0.3% NaCl and 6.5% ZnCl2301 resin anion (R.A.)s and 116 sun are passed through successively with the flow velocity of 15mL/min afterwards
It is filtered after ion exchange resin, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) 8% dehydrated alcohol, 2% crystal seed is added in step (4) filtrate three times obtained, it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(6) the wet crystallization for obtaining step (5) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 88.1%, turbidity 0.09.
Comparative example 5
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.6%
Acid, 0.3% NaCl after successively carried out after 301 resin anion (R.A.)s and 116 resin cations with the flow velocity of 15mL/min
Filter, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) after addition quality accounts for 70% ethyl alcohol of filtrate gross mass 10% three times in step (4) filtrate three times obtained
1 hour is stood, filtering obtains four filtrates；
(6) 8% dehydrated alcohol, 2% crystal seed are added in four filtrates made from step (5), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity and Turbidity measurement, purity 89.2%, turbidity 0.08.
Comparative example 6
Specific step is as follows：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, drop
Then carbohydrase is added in temperature, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time
10h then heats to 95 DEG C, keeps the temperature 20 minutes, and trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid is added into trehalose crude liquid made from step (1), is taken off
Color 45 minutes, filtering obtained first-time filtrate；
(3) step (2) it is obtained obtain adding in first-time filtrate quality account for the NaCl of first-time filtrate gross mass 0.3% with
And 6.5% ZnCl2It was successively carried out after 301 resin anion (R.A.)s and 116 resin cations with the flow velocity of 15mL/min afterwards
Filter, obtains secondary filtrate；
(4) active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is taken off
It filters, decolourizes 45 minutes after color, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.6% three times is added in step (4) filtrate three times obtained2CO3It is quiet afterwards
It sets 1 hour, filters, obtain four filtrates；
(6) 8% dehydrated alcohol, 2% crystal seed are added in four filtrates made from step (5), it is dense in 50 DEG C of progress
25 DEG C of progress spontaneous nucleations are cooled to after shrinking crystalline substance, after crystallizing 60h, 16min is centrifuged under revolving speed 2800r/min and obtains wet crystallization；
(7) the wet crystallization for obtaining step (6) dry 8h in 80 DEG C of baking oven, obtains trehalose finished product.
Obtained trehalose finished product is subjected to purity detecting, purity 89.5%, turbidity 0.09.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for reducing trehalose turbidity, which is characterized in that the method comprises the following steps：
(1) 80~100 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 10~30 points
Then carbohydrase is added in clock, cooling, carbohydrase additional amount is 12~24U/ml, adjusts pH 4.0~7.0, and control saccharification temperature is
60~65 DEG C, 8~12h of enzymolysis time, 80~100 DEG C are then heated to, keeps the temperature 10~30 minutes, it is thick that trehalose is made in filtering
Liquid；
(2) activity that quality accounts for trehalose crude liquid gross mass 0.5%~3% is added into trehalose crude liquid made from step (1)
Charcoal decolourizes 20~60 minutes, and filtering obtains first-time filtrate；
(3) in step (2) lemon obtained for obtaining addition quality in first-time filtrate and accounting for first-time filtrate gross mass 0.5~0.8%
Acid, 0.1~0.5% NaCl and 6~8% ZnCl2Successively pass through weak anion resin and Subacidity cation afterwards
It is filtered after resin, obtains secondary filtrate；
(4) be added in the secondary filtrate made from step (3) quality account for the active carbon of secondary filtrate gross mass 0.5%~3% into
It filters, decolourizes 20~60 minutes after row decoloration, filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.3~0.8% three times is added in step (4) filtrate three times obtained2CO3, 8~
0.5~1.5 hour is stood after 12% ethyl alcohol, filters, obtains four filtrates；
(6) quality is added in four filtrates made from step (5) and accounts for the dehydrated alcohol of four filtrate gross masses 5~10%, 0.1
~3% crystal seed is simultaneously cooled to 20~30 DEG C of progress spontaneous nucleations after carrying out concentration graining, after centrifugation wet crystallization；
(7) the wet crystallization that step (6) obtains is dried, obtains trehalose finished product.
2. a kind of method for reducing trehalose turbidity as described in claim 1, which is characterized in that the method includes following step
Suddenly：
(1) 85 DEG C will be heated to using trehalase reaction solution made from trehalose synthase conversion method, and keep the temperature 20 minutes, cooling,
Then carbohydrase is added, carbohydrase additional amount is 20U/ml, adjusts pH 6.0, and control saccharification temperature is 65 DEG C, enzymolysis time 10h,
95 DEG C are then heated to, keeps the temperature 20 minutes, trehalose crude liquid is made in filtering；
(2) active carbon that quality accounts for the total matter 1% of trehalose crude liquid, decoloration 45 are added into trehalose crude liquid made from step (1)
Minute, filtering obtains first-time filtrate；
(3) step (2) it is obtained obtain adding in first-time filtrate quality account for first-time filtrate gross mass 0.6% citric acid,
The ZnCl of 0.3% NaCl and 6.5%2It is successively carried out after weak anion resin and acidulous cation resin afterwards
Filtering, obtains secondary filtrate；
(4) after the active carbon that addition quality accounts for secondary filtrate gross mass 1% in the secondary filtrate made from step (3) is decolourized
Filtering is decolourized 45 minutes, and filtering obtains filtrate three times；
(5) Na that quality accounts for filtrate gross mass 0.6% three times is added in step (4) filtrate three times obtained2CO3, 10% second
1 hour is stood after alcohol, filters, obtains four filtrates；
(6) crystalline substance that quality accounts for the dehydrated alcohol of four filtrate gross masses 8%, 2% is added in four filtrates made from step (5)
It plants and is cooled to 25 DEG C of progress spontaneous nucleations after carrying out concentration graining, wet crystallization is obtained after centrifugation；
(7) the wet crystallization that step (6) obtains is dried, obtains trehalose finished product.
3. a kind of method for reducing trehalose turbidity as claimed in claim 1 or 2, which is characterized in that in the step (3),
First-time filtrate is 15~20mL/min followed by the speed of weak anion resin and acidulous cation resin.
4. a kind of method for reducing trehalose turbidity a method according to any one of claims 1-3, which is characterized in that the step (3)
In, first-time filtrate is 15mL/min followed by the speed of weak anion resin and acidulous cation resin.
5. a kind of method of reduction trehalose turbidity as described in claim 1-4 is any, which is characterized in that the step (6)
In, the temperature of concentration is 45~55 DEG C.
6. a kind of method for reducing trehalose turbidity a method as claimed in any one of claims 1 to 5, which is characterized in that the step (6)
In, the time of crystallization is 60~70h.
7. a kind of method of reduction trehalose turbidity as described in claim 1-6 is any, which is characterized in that the step (6)
In, the time of centrifugation is 10~20min.
8. a kind of method for reducing trehalose turbidity as claimed in claim 1, which is characterized in that the step (6)
In, 2500~3500r/min of revolving speed of centrifugation.
9. a kind of method for reducing trehalose turbidity a method as claimed in any one of claims 1-8, which is characterized in that the step (7)
In drying be by wet crystallization in 75~85 DEG C of baking oven dry 6~10h.
10. a kind of application of the method for any reduction trehalose turbidity of claim 1-9 in terms of preparing trehalose.
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