CN105925642B - With the method for microbe fermentation method industrialized production trehalose - Google Patents
With the method for microbe fermentation method industrialized production trehalose Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
With the method for microbe fermentation method industrialized production trehalose, include the following steps: that hydrolysis of trehalose enzyme and trehalose generate fermentation, starch liquefacation, catalysis reaction, the saccharification reaction, decolorization filtering, ion exchange, concentration, crystallization, centrifugation, drying, UF membrane of enzyme (X/Y).Beneficial effects of the present invention: 1, the present invention is made from starch, enzyme law catalysis produce trehalose, enable trehalose largely, inexpensively carry out industry law production.2, enzyme activity is high, and dosage is few, reduces production cost.3, the trehalose mother liquor being centrifuged is improved by UF membrane, content of trehalose, can be returned with crystallization, be further increased trehalose yield.4, the trehalose crystallization processes innovated, content of trehalose is 50% or more, and glucose content is 20% hereinafter, primary crystallization trehalose finished product content is up to 98.5% or more.5, have great advantage compared with maltose conversion Technology of Trehalose, high income, production cost is low.
Description
Technical field
The invention belongs to starch sugar production technical fields, and in particular to a kind of to use microbe fermentation method industrialized production seaweed
The method of sugar.
Background technique
In November, 2000 the FAO (Food and Agriculture Organization of the United Nation) and World Health Organization (FAO/WHO) food additives joint specialist committee member
Meeting (JECFA) confirms that trehalose as texturizer, stabilizer, moisturizer, the function of sweetener, is permitted the daily of trehalose
Perhaps intake is with no restriction；In March, 2005, it is new energy food that the Ministry of Public Health, China, which ratifies trehalose,.Trehalose production becomes heat
The exploration project of door, production method cause the extensive concern of domestic and international starch sugar manufacturing enterprise.Production method that there are many reports,
Can industrialized production, low cost production method it is also seldom.
Trehalose is freezing, and plays under the harsh environment such as drying, hyperosmosis organism film, memebrane protein, DNA etc.
Protection effect, preservation and regeneration with life have very deep relationship, are the natural materials that pole is gazed at, have been widely used now
In food, medicine, cosmetic field, the application study with people to trehalose exclusive function and characteristic, purposes are opened later
Hair will will more be extended to more deep and broad development, application field, will be that dosage is bigger in starchy carbohydrate, application prospect is more wide
Sugar products of new generation.
General preparation method (microorganism extraction process, microbe fermentation method, the enzyme process manufacture, gene for the trehalose having been reported that at present
Recombination method) it is all small test or theory, industrialized production can not be carried out, and cost is very high.Wheat is used there are also a kind of
The industrialized preparing process of bud sugar conversion, but yield is low, high production cost.
Summary of the invention
It is an object of that present invention to provide a kind of methods with microbe fermentation method industrialized production trehalose.
The purpose of the present invention is achieved through the following technical solutions:
With the method for microbe fermentation method industrialized production trehalose, include the following steps:
(1) hydrolysis of trehalose enzyme and trehalose generate the fermentation of enzyme (X/Y): passing through gene constructed obtained recombination large intestine bar
Bacterium strain cultivates host strain using LB culture medium, including two steps:
1. the activation and preparation of seed: above-mentioned recombination bacillus coli strain is connected in LB culture medium, 37 DEG C of culture 12h,
Obtain shake-flask seed liquid；The LB culture medium includes: bacteriological peptone 10g/L, yeast powder 5g/L, Nacl10g/L；Adjust PH
It is worth 7.4,121 DEG C of sterilizing 20min；
2. upper tank fermentation: shake-flask seed liquid being seeded in the fermentor equipped with TB culture medium, 37 DEG C of cultures to OD(bacterium
Growing state) grow to 0.6, inducer lactose is added, so that lactose concn is reached 4mg/ml, then in 32 DEG C of Fiber differentiations 24-
36h obtains the fermentation liquid containing X/Y enzyme；Fermentation liquid breaks cell wall through high pressure homogenizer twice, and then barrel filling refrigeration is standby
With；The TB culture medium is made of peptone 12g, yeast extract 24g, glycerol 4ml, high pressure sterilization after each component dissolution, cold
But to 60 DEG C, then plus 100ml sterilizing 170mol/L potassium dihydrogen phosphate and 0.72mol/L dipotassium hydrogen phosphate solution；
(2) starch liquefacation: matching starch slurry concentration 14--15Be, and pH value is transferred to 5.0-6.0, adds fire resistant alpha-diastase
400ml/ tons of starch；One time 105-108 DEG C of injection temperation, 20-40 minutes are kept the temperature, after iodine examination reaction is qualified, then secondary injection
130-135 DEG C of enzyme-removal temperature, it is produced into liquefier；44-46 DEG C are cooled to by plate heat exchanger, into saccharifying tank catalysis, sugar
Change；
(3) catalysis reaction: 44-46 DEG C of liquefier temperature is mixed up in saccharifying tank, every cube of liquefier adds Pullulanase
200ml, trehalose generate enzyme Y enzyme 7000ml(enzyme fermentation liquid), hydrolysis of trehalose enzyme 2000ml (enzyme fermentation liquid)；Heat preservation catalysis is anti-
It answers 30--36 hours, generates liquid glucose；
(4) saccharification reaction: the liquid glucose after catalysis reaction is warming up to 62 DEG C, with hydrochloric acid tune every cube of liquid glucose of PH4.2-4.5.
Add compounded saccharifying enzyme 250ml. heat preservation saccharification 24 hours, after detection dextrin reaction is qualified, is warming up to 80--85 DEG C of enzyme deactivation, generates sugar
Change liquid；If dextrin reaction is unqualified, carbohydrase is added, extends saccharificatinn period enzyme deactivation after qualification；
(5) decolorization filtering: the saccharified liquid after enzyme deactivation is sent to bleacher, by 3% plus active carbon, 2% of saccharified liquid containing granular type object
Add pearlite filtering aid, decoloration stirring 30 minutes, limpid, the transparent, foreign that flows back is sent to basin before ion exchange；
(6) ion exchange: filtered saccharified liquid passes sequentially through the sun regenerated -- negative -- sun -- anion-exchange column, elder generation
Stream is returned, flow is controlled, conductivity is less than 20us/cm, and PH is greater than 4.5, is sent to tank before concentration；
(7) be concentrated: the good saccharified liquid of ion exchange is concentrated to concentration 72% -73% with quadruple effect inspissator, adjusts before being sent to crystallization
Dense tank, is produced into concentrated sugar；
(8) crystallize: concentrated sugar is cooled down by plate-type heat-exchange into crystallizer, and temperature controls 46-50 DEG C, is added by the 3% of total weight
Dry crystal seed, growing the grain 4-8 hours, temperature lowering curve control was dropped to 16 DEG C for 60 hours from 46 DEG C, 46-30 DEG C of high-temperature region temperature drop
Slowly, 30-16 DEG C of low-temperature space drops are fast；
(9) it is centrifuged: using upper suspension mechanical discharging automatic centrifuge, the sugared cake of advantages of good crystallization is pumped on centrifuge with rotor
Distributing trough opens centrifuge centrifugation, and the distilled water with temperature lower than 20 DEG C presses 5% affination of sugared cake amount, and detection content of trehalose is big
In 98.5%, if content not enough use again more than method affination it is primary, it is qualified until reaching；
(10) dry:, a hot blast temperature control to 65-dry with boiling fluidised-bed drying machine or pneumatic conveying drying unit
75 DEG C, the control of secondary hot-air temperature is to 75-85 DEG C, and cold wind temperature less than 20 DEG C, pack less than 37 DEG C by commerieal sugar temperature；
(11) UF membrane: the mother liquor after centrifugation contains glucose 21%, contains trehalose 20%, the organic molecule passed through using particular bore
Film separates the glucose in mother liquor with trehalose；Content of trehalose, which can return to be fitted in ion exchange liquid greater than 80%, is concentrated knot
Crystalline substance, glucose content are greater than 95% and can be used for producing fructose syrup.
Beneficial effects of the present invention: 1, the present invention is made from starch, and enzyme law catalysis produces trehalose, enables trehalose
Largely, industry law production, and the promotion and development very big to starch industry are inexpensively carried out.2, enzyme activity is high, and dosage is few,
And it can be used directly to catalytic production with fermentation liquid, reduce production cost.3, the trehalose mother liquor being centrifuged passes through UF membrane,
Content of trehalose improves, and can return with crystallization, further increase trehalose yield.4, the trehalose crystallization processes innovated, pass through
Saccharified liquid after catalysis reaction, by refining steps such as decoloration, ion exchanges, content of trehalose is 50% or more, glucose content
20% hereinafter, by the reasonable cerium sulphate crystal index of setting, the important indicators such as temperature lowering curve, sugar entering saturation degree, sugar entering temperature,
Primary crystallization trehalose finished product content is up to 98.5% or more.5, have compared with maltose conversion Technology of Trehalose very big excellent
Gesture, high income, production cost are low.
Specific embodiment
The present invention can be described in more detail in the following examples, but the invention is not limited in any way.
Embodiment 1:
Cornstarch 1200kg is weighed, pH value 5.68, starch slurry concentration 14Be, an injection temperation are added water and stirred and adjust
106 DEG C, 40 minutes are kept the temperature, checks that iodine colour response is qualified, 135 DEG C of secondary injection temperature；Obtain liquefier 4.06M3, concentration
23.6Bx, liquefy DE value 9.83%；46 DEG C are cooled to, Pullulanase 800ml is added, trehalose is added to generate enzyme Y enzyme 28kg(enzyme fermentation
Liquid), hydrolysis of trehalose enzyme Z enzyme 8kg(enzyme fermentation liquid), catalysis reaction 30h；61 DEG C are warming up to, with hydrochloric acid tune PH4.4, sugaring
Enzyme 400ml, saccharification reaction for 24 hours, check that dextrin reaction is qualified；It is warming up to 85 DEG C of enzyme deactivations and keeps the temperature 30 minutes, add active carbon decoloring mistake
Filter, obtains concentration 23.5%, the decoloration clear liquid of conductivity 1365us/cm, volume 3.97M3；Pass through cation and anion exchange, conductivity
Drop to 21us/cm, PH5.8；It is concentrated to concentration 72.2%, volume 1.03M3, content of trehalose 71.22%, glucose content
28.10%；In 1.2M3 crystallizer, add crystal seed 3kg, temperature drops to 16 DEG C from 48 DEG C；Centrifugation obtains damp sugar 480kg.With boiling
Fluid bed dryer or pneumatic conveying drying unit are dry, and to 65-75 DEG C, the control of secondary hot-air temperature is arrived for a hot blast temperature control
75-85 DEG C, cold wind temperature less than 20 DEG C, pack less than 37 DEG C by commerieal sugar temperature；The trehalose mother liquor that will be centrifuged out, with film point
From rear, one kind containing glucose 95.5%, is produced into fructose syrup, and another kind contains trehalose 78.5%, comes with preceding workshop section's ion exchange
Sugar is returned to be fitted on to be concentrated and be crystallized again, concentration 72.5%, volume 1.10M3, content of trehalose 75.12%, glucose content
24.21%, in 1.2M3 crystallizer plus crystal seed 3kg, temperature drop to 16 degree from 48 degree, and centrifugation obtains damp sugar 502kg.
It is as shown in the table by the crystalline trehalose national standard testing result of GB/T23529-2009:
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (1)
1. with the method for microbe fermentation method industrialized production trehalose, which comprises the steps of:
(1) hydrolysis of trehalose enzyme and trehalose generate the fermentation of enzyme: by gene constructed obtained recombination bacillus coli strain, adopting
Host strain is cultivated with LB culture medium, including two steps:
1. the activation and preparation of seed: above-mentioned recombination bacillus coli strain being connected in LB culture medium, 37 DEG C of culture 12h, obtained
Shake-flask seed liquid；The LB culture medium includes: bacteriological peptone 10g/L, yeast powder 5g/L, Nacl10g/L；PH value is adjusted to arrive
7.4,121 DEG C of sterilizing 20min；
2. upper tank fermentation: shake-flask seed liquid being seeded in the fermentor equipped with TB culture medium, 37 DEG C of cultures to bacterium OD are arrived
0.6, inducer lactose is added, lactose concn is made to reach 4mg/ml, then in 32 DEG C of 24-36h of Fiber differentiation, obtains containing sea
Algae glycosylhydrolase and trehalose generate the fermentation liquid of enzyme；Fermentation liquid breaks bacteria cell wall through high pressure homogenizer twice, then fills
Bucket refrigeration is spare；The TB culture medium is made of peptone 12g, yeast extract 24g, glycerol 4ml, high pressure after each component dissolution
Sterilizing, is cooled to 60 DEG C, then plus 100ml sterilizing 170mol/L potassium dihydrogen phosphate and 0.72mol/L dipotassium hydrogen phosphate it is molten
Liquid；
(2) starch liquefacation: matching starch slurry concentration 14--15Be, and pH value is transferred to 5.0-6.0, adds 400ml/ tons of fire resistant alpha-diastase
Starch；One time 105-108 DEG C of injection temperation, 20-40 minutes are kept the temperature, after iodine examination reaction is qualified, then secondary injection enzyme-removal temperature
130-135 DEG C, it is produced into liquefier；44-46 DEG C are cooled to by plate heat exchanger, into saccharifying tank catalysis, saccharification；
(3) catalysis reaction: mixing up 44-46 DEG C of liquefier temperature in saccharifying tank, and every cube of liquefier adds Pullulanase 200ml,
Trehalose generates enzyme 7000ml, hydrolysis of trehalose enzyme 2000ml；Heat preservation catalysis reaction 30--36 hours, generate liquid glucose；
(4) saccharification reaction: the liquid glucose after catalysis reaction is warming up to 62 DEG C, and with hydrochloric acid tune PH4.2-4.5, every cube of liquid glucose adds multiple
Carbohydrase 250ml is closed, heat preservation saccharification 24 hours is warming up to 80--85 DEG C of enzyme deactivation after detection dextrin reaction is qualified, generates saccharification
Liquid；If dextrin reaction is unqualified, carbohydrase is added, extends saccharificatinn period enzyme deactivation after qualification；
(5) decolorization filtering: the saccharified liquid after enzyme deactivation is sent to bleacher, by 3% plus active carbon of saccharified liquid containing granular type object, 2% plus treasure
Pearl rock filter aid, decoloration stirring 30 minutes, limpid, the transparent, foreign that flows back are sent to basin before ion exchange；
(6) ion exchange: filtered saccharified liquid passes sequentially through the sun regenerated, and -- negative -- sun -- anion-exchange column is first returned
Stream controls flow, and conductivity is less than 20us/cm, and PH is greater than 4.5, is sent to tank before concentration；
(7) be concentrated: the good saccharified liquid of ion exchange is concentrated to concentration 72% -73% with quadruple effect inspissator, thickens before being sent to crystallization
Tank is produced into concentrated sugar；
(8) crystallize: concentrated sugar is cooled down by plate-type heat-exchange into crystallizer, and temperature controls 46-50 DEG C, by 3% plus dry crystalline substance of total weight
Kind, growing the grain 4-8 hours, temperature lowering curve control was dropped to 16 DEG C for 60 hours from 46 DEG C, and 46-30 DEG C of high-temperature region temperature slows down, low
30-16 DEG C of warm area drops are fast；
(9) it is centrifuged: using upper suspension mechanical discharging automatic centrifuge, the sugared cake of advantages of good crystallization is pumped on centrifuge with rotor to be distributed
Slot opens centrifuge centrifugation, and the distilled water with temperature lower than 20 DEG C presses 5% affination of sugared cake amount, and detection content of trehalose is greater than
98.5%, if content not enough use again more than method affination it is primary, until reaching qualified；
(10) dry: dry with boiling fluidised-bed drying machine or pneumatic conveying drying unit, a hot blast temperature is controlled to 65-75 DEG C,
The control of secondary hot-air temperature is to 75-85 DEG C, and cold wind temperature less than 20 DEG C, pack less than 37 DEG C by commerieal sugar temperature；
(11) UF membrane: mother liquor after centrifugation contains glucose 21%, contains trehalose 20%, using the organic molecular film of special pore size distribution,
Glucose in mother liquor is separated with trehalose；Content of trehalose can return greater than 80% and be fitted on condensing crystallizing in ion exchange liquid, Portugal
Grape sugared content is greater than 95% and can be used for producing fructose syrup.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107739745A (en) * | 2017-10-10 | 2018-02-27 | 溧阳维信生物科技有限公司 | α, the method for alpha trehalose dihydrate are prepared using membrane separation technique |
| CN107488689B (en) * | 2017-10-10 | 2021-04-27 | 溧阳维信生物科技有限公司 | Method for preparing alpha, alpha-trehalose dihydrate by adopting electrodialysis technology |
| CN108130351B (en) * | 2018-01-26 | 2021-08-06 | 通辽梅花生物科技有限公司 | Preparation method of large-particle trehalose |
| CN108588148A (en) * | 2018-07-06 | 2018-09-28 | 安徽民祯生物工程有限公司 | A kind of method of rice fermentation production trehalose |
| CN108624637B (en) * | 2018-08-24 | 2021-10-19 | 湖南汇升生物科技有限公司 | Method for producing trehalose by using rice |
| CN109251952A (en) * | 2018-09-18 | 2019-01-22 | 溧阳维信生物科技有限公司 | A method of preparing 79-87% a, a-trehalose dihydrate |
| CN110055293B (en) * | 2019-04-29 | 2020-10-27 | 郑州谦益食品有限公司 | Trehalose synthesis method |
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