CN106947796A - A kind of D trehaloses purifying technique - Google Patents
A kind of D trehaloses purifying technique Download PDFInfo
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- CN106947796A CN106947796A CN201710221252.0A CN201710221252A CN106947796A CN 106947796 A CN106947796 A CN 106947796A CN 201710221252 A CN201710221252 A CN 201710221252A CN 106947796 A CN106947796 A CN 106947796A
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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
- C12P19/14—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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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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Abstract
The invention belongs to the extractive technique field of algal polysaccharides there is provided a kind of D trehaloses purifying technique, first by Feedstock treating, slurries are made；Acidic cellulase is added into slurries；It is subsequently added into alkaline pectase；Centrifuge removal of impurities, obtains enzymolysis liquid；Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially passes through the milipore filter that the milipore filter and molecular cut off that molecular cut off is 300 are 400, the D trehaloses for obtaining molecular weight between 300 400 purify solution；D trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D trehaloses is separated out completely from solution；Filtering, makes D trehaloses be separated from the mixed solution of D trehaloses purification solution and absolute ethyl alcohol；Drying process, the D trehaloses purified.D trehaloses recovery rate of the present invention is high, and more general method of purification improves 4 to 7 percentage points.
Description
Technical field
The invention belongs to the extractive technique field of algal polysaccharides, in particular it relates to a kind of D- trehaloses purifying technique.
Background technology
Trehalose, namely D- trehaloses, are a kind of safe and reliable natural carbohydrates, and 1832 by Wiggers by it from black
Extracted first in the ergot of wheat, subsequent research finds trehalose many edible animal and plant and micro- life in nature
All it is widely present in object, edible mushroom excrement, seaweeds, beans shrimp, bread, beer tall building yeast hair in such as people's daily life
There is the higher trehalose of content in ferment food.
Trehalose has anti-oxidant, anticoagulation, antitumor, antiviral, suppression complement activation and absorbs the functions such as heavy metal,
In addition, because with safety, reliable, environmentally friendly characteristic uses it for the production of veterinary drug with higher market prospects.
In the prior art, general to extract concentration fern amylose using Vacuum Heat method for concentration, this method easily makes fern fiber crops
The antioxidation activity reduction of polysaccharide, reduces its medical value, in addition, only slightly being carried to fern amylose in existing method
It is pure, it is impure more in the thick fern amylose purified out, it is unfavorable for the development and application of trehalose.
Current trehalose preparation method mainly has microorganism extraction process, fermentation method etc..
Microorganism extraction process high cost, extraction source is limited, largely governs trehalose large-scale industrial production.
Fermentation method conversion ratio is low, and fermentation broth contents are complicated, the extracting of trehalose, refined difficulty, thus reduction is produced into
Originally it is, very unfavorable in terms of promotion popularization and application.
CN200610016527.9 discloses a kind of isolation and purification method of trehalose, comprises the concrete steps that：To every gram of marine alga
The 0.14-0.28 enzyme unit that is saccharified is added in carbohydrase reaction solution, 6-12h is hydrolyzed under the conditions of 50-60 DEG C, pH3.5-5.5, then to
The saccharomyces cerevisiae of 3-10% weight is inoculated with carbohydrase enzymolysis liquid, 6-16h is cultivated under the conditions of 25-30 DEG C, pH5.5-7.5, is passivated
The centrifugation of after fermentation liquid, ultrafiltration, ion exchange, concentration, crystallizing and drying obtain trehalose.But this method excessive cycle, trehalose yield
Comparison of ingredients is complicated in not high, later stage fermentation liquid, and separation also acquires a certain degree of difficulty, and is not suitable for industrial production.
Therefore, how to develop it is a kind of improve the Methods of Fucoidan of above-mentioned technological deficiency, be correlative technology field
Urgent problem.
The content of the invention
For defect of the prior art, it is an object of the invention to provide a kind of D- trehaloses purifying technique.
A kind of D- trehaloses purifying technique that the present invention is provided, solves the D- trehaloses that method of the prior art is extracted
Purity is low, the problem of D- trehalose recovery rates are low.
A kind of D- trehaloses purifying technique provided according to the present invention, comprises the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 3-5 times, drain, shred, with the deionized water soaking at room temperature of 6-8 times of sea-tangle weight
1-2 days, mashing processing is carried out after the completion of immersion, slurries are made；
Step（2）：Acidic cellulase is added into slurries, 2-6h is digested in the water-bath for being placed in 40-54 DEG C, PH controls exist
Stirring is not stopped between 4.5-6.5, in enzymolysis process；
Step（3）：Alkaline pectase is added, 2-6h is digested in the water-bath for being placed in 50-65 DEG C, PH is controlled between 8.0-10.0,
Stirring is not stopped in enzymolysis process；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be kept completely separate out from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
Preferably, the step（5）Before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution,
Cleaned again with clear water, the pH value of the milipore filter is reached 7.0-7.4.
Preferably, the step（7）Described in D- trehaloses purification solution and absolute ethyl alcohol volume ratio be 1：4-5.
Preferably, the step（4）Middle centrifuge speed is 1000-1200r/min, and centrifugation time is 20-30min.
Preferably, the filter membrane used in the process of ultrafiltration treatment is Kynoar filter membrane.
Preferably, the acidic cellulase is decomposed by trichoderma reesei 91-3 bacterial strains and obtained.
Preferably, the trichoderma reesei 91-3 bacterial strains reduce sourness cellulase method for first trichoderma reesei A3 carried out
After ultraviolet and nitrosoguanidine complex mutation, by treated spore inoculating on fiber double-layer plate, trained under the conditions of 30 DEG C
Support 5-8 days, placed 7-10 days under the conditions of 15 DEG C, select transparent loop diameter and the larger single bacterium colony of colony diameter ratio carries out triangle
Bottle solid state fermentation is screened again, obtains trichoderma reesei 91-3 bacterial strains.
Preferably, the step（8）Drying temperature is 60-80 DEG C.
Compared with prior art, the present invention has following beneficial effect：
（1）The characteristic of the present invention includes two parts, is on the one hand that the stage is discharged from sea-tangle cell membrane in trehalose, the present invention
Cell membrane is carried out with acidic cellulase and alkaline pectase it is discrete, because enzyme has unicity, cellulase and pectase
The main constituents cellulose and pectic substance of cell membrane can be decomposed, so as to by cell wall damage, make trehalose therein
Discharge, in this process because present invention selection is in acidic cellulase and alkaline pectase most suitable pH condition and temperature
Degree condition is carried out, and the activity of two kinds of enzymes reaches maximum, as a result makes cellulose and pectic substance complete decomposition, and cell membrane is discrete completely,
Finally trehalose is set fully to discharge；On the other hand, due in sea-tangle cell membrane in addition to also having trehalose also containing other carbohydrates,
The compositions such as protein, inorganic matter, it is therefore necessary to which the higher trehalose of purity, marine alga can just be obtained by further being separated
The molecular weight of sugar is 320, therefore the present invention uses hyperfiltration process, using different milipore filters, accurately separates trehalose
Out.Using the purifying technique of the present invention the more general method of purification of purification rate can be made to improve 4 to 7 percentage points；
（2）The present invention unused strong acid, highly basic and poisonous organic solvent, have accomplished institute in clean environment firendly, and the inventive method
The waste liquid and waste residue of generation can be used to produce yeast extract, almost without discharge；
（3）The D- trehaloses that the present invention is extracted have anti-oxidant, anticoagulation, antitumor, antiviral, suppression complement activation and absorption
Heavy metal etc. is acted on, therefore available for the preparation of veterinary drug.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.
A kind of D- trehaloses purifying technique that the present invention is provided, solves the D- trehaloses that method of the prior art is extracted
Purity is low, the problem of D- trehalose recovery rates are low.
A kind of D- trehaloses purifying technique provided according to the present invention, comprises the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 3-5 times, drain, shred, with the deionized water soaking at room temperature of 6-8 times of sea-tangle weight
1-2 days, mashing processing is carried out after the completion of immersion, slurries are made.The purpose wherein soaked is in order that sea-tangle cell expansion, makes thin
Various composition in cell wall is in active state, beneficial to enzyme digestion reaction and separating-purifying；
Step（2）：Acidic cellulase is added into slurries, 2-6h is digested in the water-bath for being placed in 40-54 DEG C, PH is controlled in 4.5-
Stirring is not stopped between 6.5, in enzymolysis process.Because PH controls are between 4.5-6.5, temperature control is when 40-54 DEG C, acid
The active highest of property cellulase, thus hydrolysis result be also it is best, can by the cellulase hydrolysis contained in cell membrane so that
Cell membrane is destroyed, and discharges fucoidin therein；
Step（3）：Alkaline pectase is added, 2-6h is digested in the water-bath for being placed in 50-65 DEG C, PH is controlled between 8.0-10.0,
Do not stop in enzymolysis process stirring, due to PH control between 8.0-10.0, temperature control when 50-65 DEG C, pectase
Active highest, therefore hydrolysis result is also best, can digest the pectic substance contained in cell membrane, so that cell membrane is destroyed,
And discharge fucoidin therein；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be separated from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
Preferably, the step（5）Before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution,
Cleaned again with clear water, the pH value of the milipore filter is reached 7.0-7.4.
Preferably, the step（7）Described in D- trehaloses purification solution and absolute ethyl alcohol volume ratio be 1：4-5.
Preferably, the step（4）Middle centrifuge speed is 1000-1200r/min, and centrifugation time is 20-30min.
Preferably, the filter membrane used in the process of ultrafiltration treatment is Kynoar filter membrane.
Preferably, the acidic cellulase is decomposed by trichoderma reesei 91-3 bacterial strains and obtained.
Preferably, the trichoderma reesei 91-3 bacterial strains reduce sourness cellulase method for first trichoderma reesei A3 carried out
After ultraviolet and nitrosoguanidine complex mutation, by treated spore inoculating on fiber double-layer plate, trained under the conditions of 30 DEG C
Support 5-8 days, placed 7-10 days under the conditions of 15 DEG C, select transparent loop diameter and the larger single bacterium colony of colony diameter ratio carries out triangle
Bottle solid state fermentation is screened again, obtains cellulase-producing vigor very high trichoderma reesei 91-3 bacterial strains.
Preferably, the step（8）Drying temperature is 60-80 DEG C.
Compared with prior art, the present invention has following beneficial effect：
（1）The characteristic of the present invention includes two parts, is on the one hand that the stage is discharged from sea-tangle cell membrane in trehalose, the present invention
Cell membrane is carried out with acidic cellulase and alkaline pectase it is discrete, because enzyme has unicity, cellulase and pectase
The main constituents cellulose and pectic substance of cell membrane can be decomposed, so as to by cell wall damage, make trehalose therein
Discharge, in this process because present invention selection is in acidic cellulase and alkaline pectase most suitable pH condition and temperature
Degree condition is carried out, and the activity of two kinds of enzymes reaches maximum, as a result makes cellulose and pectic substance complete decomposition, and cell membrane is discrete completely,
Finally trehalose is set fully to discharge；On the other hand, due in sea-tangle cell membrane in addition to also having trehalose also containing other carbohydrates,
The compositions such as protein, inorganic matter, it is therefore necessary to which the higher trehalose of purity, marine alga can just be obtained by further being separated
The molecular weight of sugar is 320, therefore the present invention uses hyperfiltration process, using different milipore filters, accurately separates trehalose
Out.Using the purifying technique of the present invention the more general method of purification of purification rate can be made to improve 4 to 7 percentage points；
（2）The present invention unused strong acid, highly basic and poisonous organic solvent, have accomplished institute in clean environment firendly, and the inventive method
The waste liquid and waste residue of generation can be used to produce yeast extract, almost without discharge；
（3）The D- trehaloses that the present invention is extracted have anti-oxidant, anticoagulation, antitumor, antiviral, suppression complement activation and absorption
Heavy metal etc. is acted on, therefore available for the preparation of veterinary drug.
Embodiment 1
A kind of D- trehaloses purifying technique that the present embodiment is provided, comprises the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 5 times, drain, shred, with the deionized water soaking at room temperature 2 days of 6 times of sea-tangle weight,
Mashing processing is carried out after the completion of immersion, slurries are made；
Step（2）：Acidic cellulase is added into slurries, 6h is digested in the water-bath for being placed in 40 DEG C, PH controls are in 4.5, enzymolysis
During do not stop stirring；
Step（3）：Alkaline pectase is added, 2h is digested in the water-bath for being placed in 65 DEG C, PH controls do not stop in 10.0, enzymolysis process
Stirring；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be separated from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
The step（5）Before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution, then use clear water
Cleaned, the pH value of the milipore filter is reached 7.0.
The step（7）Described in D- trehaloses purification solution and absolute ethyl alcohol volume ratio be 1：5.
The step（4）Middle centrifuge speed is 1000r/min, and centrifugation time is 30min.
The filter membrane used in the process of ultrafiltration treatment is Kynoar filter membrane.
The acidic cellulase is decomposed by trichoderma reesei 91-3 bacterial strains and obtained.
The trichoderma reesei 91-3 bacterial strains reduce sourness cellulase method for first by trichoderma reesei A3 carry out ultraviolet
After nitrosoguanidine complex mutation, by treated spore inoculating on fiber double-layer plate, cultivated 8 days under the conditions of 30 DEG C,
Placed 7 days under the conditions of 15 DEG C, select transparent loop diameter and the larger single bacterium colony of colony diameter ratio carries out triangular flask solid state fermentation
Screen again, obtain trichoderma reesei 91-3 bacterial strains.
The step（8）Drying temperature is 80 DEG C.
The purifying technique of the present embodiment can make the more general method of purification of purification rate improve 5 percentage points.
Embodiment 2
A kind of D- trehaloses purifying technique that the present embodiment is provided, comprises the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 3 times, drain, shred, with the deionized water soaking at room temperature 1 day of 8 times of sea-tangle weight,
Mashing processing is carried out after the completion of immersion, slurries are made；
Step（2）：Acidic cellulase is added into slurries, 2h is digested in the water-bath for being placed in 54 DEG C, PH controls are in 6.5, enzymolysis
During do not stop stirring；
Step（3）：Alkaline pectase is added, 6h is digested in the water-bath for being placed in 50 DEG C, PH controls do not stop in 8.0, enzymolysis process
Stirring；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be separated from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
The step（5）Before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution, then use clear water
Cleaned, the pH value of the milipore filter is reached 7.0.
The step（7）Described in D- trehaloses purification solution and absolute ethyl alcohol volume ratio be 1：4.
The step（4）Middle centrifuge speed is 1200r/min, and centrifugation time is 20min.
The filter membrane used in the process of ultrafiltration treatment is Kynoar filter membrane.
The acidic cellulase is decomposed by trichoderma reesei 91-3 bacterial strains and obtained.
The trichoderma reesei 91-3 bacterial strains reduce sourness cellulase method for first by trichoderma reesei A3 carry out ultraviolet
After nitrosoguanidine complex mutation, by treated spore inoculating on fiber double-layer plate, cultivated 5 days under the conditions of 30 DEG C,
Placed 10 days under the conditions of 15 DEG C, select transparent loop diameter and the larger single bacterium colony of colony diameter ratio carries out triangular flask solid state fermentation
Screen again, obtain trichoderma reesei 91-3 bacterial strains.
The step（8）Drying temperature is 60 DEG C.
The purifying technique of the present embodiment can make the more general method of purification of purification rate improve 7 percentage points.
Embodiment 3
A kind of D- trehaloses purifying technique that the present embodiment is provided, comprises the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 4 times, drain, shred, with the deionized water soaking at room temperature 1 day of 7 times of sea-tangle weight,
Mashing processing is carried out after the completion of immersion, slurries are made；
Step（2）：Acidic cellulase is added into slurries, 4h is digested in the water-bath for being placed in 45 DEG C, PH controls are in 5.0, enzymolysis
During do not stop stirring；
Step（3）：Alkaline pectase is added, 4h is digested in the water-bath for being placed in 55 DEG C, PH controls do not stop in 9.0, enzymolysis process
Stirring；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be separated from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
The step（5）Before hyperfiltration treatment is carried out, the milipore filter is first cleaned with sodium hydroxide solution, then use clear water
Cleaned, the pH value of the milipore filter is reached 7.2.
The step（7）Described in D- trehaloses purification solution and absolute ethyl alcohol volume ratio be 1：4.
The step（4）Middle centrifuge speed is 1100r/min, and centrifugation time is 25min.
The filter membrane used in the process of ultrafiltration treatment is Kynoar filter membrane.
The acidic cellulase is decomposed by trichoderma reesei 91-3 bacterial strains and obtained.
The trichoderma reesei 91-3 bacterial strains reduce sourness cellulase method for first by trichoderma reesei A3 carry out ultraviolet
After nitrosoguanidine complex mutation, by treated spore inoculating on fiber double-layer plate, cultivated 6 days under the conditions of 30 DEG C,
Placed 8 days under the conditions of 15 DEG C, select transparent loop diameter and the larger single bacterium colony of colony diameter ratio carries out triangular flask solid state fermentation
Screen again, obtain trichoderma reesei 91-3 bacterial strains.
The step（8）Drying temperature is 70 DEG C.
The purifying technique of the present embodiment can make the more general method of purification of purification rate improve 4 percentage points.
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substance of the present invention.
Claims (8)
1. a kind of D- trehaloses purifying technique, it is characterised in that comprise the following steps：
Step（1）：Take Fresh Laminaria Japonica to clean 3-5 times, drain, shred, with the deionized water soaking at room temperature of 6-8 times of sea-tangle weight
1-2 days, mashing processing is carried out after the completion of immersion, slurries are made；
Step（2）：Acidic cellulase is added into slurries, 2-6h is digested in the water-bath for being placed in 40-54 DEG C, PH is controlled in 4.5-
6.5, do not stop stirring in enzymolysis process；
Step（3）：Alkaline pectase is added, 2-6h is digested in the water-bath for being placed in 50-65 DEG C, PH is controlled between 8.0-10.0,
Stirring is not stopped in enzymolysis process；
Step（4）：Centrifuge removal of impurities, obtains enzymolysis liquid；
Step（5）：Enzymolysis liquid is subjected to hyperfiltration treatment by milipore filter, enzymolysis liquid sequentially pass through molecular cut off for 300 it is super
Filter membrane and the milipore filter that molecular cut off is 400, the D- trehaloses for obtaining molecular weight between 300-400 purify solution；
Step（6）：D- trehaloses purification solution is dehydrated with absolute ethyl alcohol mixing, D- trehaloses is analysed completely from solution
Go out；
Step（7）：D- trehaloses are made to be kept completely separate out from the mixed solution of D- trehaloses purification solution and absolute ethyl alcohol；
Step（8）：It is dried to obtain the D- trehaloses of purification.
2. D- trehaloses purifying technique according to claim 1, it is characterised in that：The step（5）Carrying out at ultrafiltration
Before reason, the milipore filter is first cleaned with sodium hydroxide solution, then cleaned with clear water, the pH value of the milipore filter is reached
7.0-7.4。
3. D- trehaloses purifying technique according to claim 1, it is characterised in that：The step（7）Described in D- marine algas
The volume ratio of sugar purification solution and absolute ethyl alcohol is 1：4-5.
4. D- trehaloses purifying technique according to claim 1, it is characterised in that：The step（4）Middle centrifuge speed
For 1000-1200r/min, centrifugation time is 20-30min.
5. D- trehaloses purifying technique according to claim 3, it is characterised in that：Used in the process of ultrafiltration treatment
Filter membrane is Kynoar filter membrane.
6. D- trehaloses purifying technique according to claim 1, it is characterised in that：The acidic cellulase is by Richter scale wood
Mould 91-3 bacterial strains are decomposed and obtained.
7. D- trehaloses purifying technique according to claim 6, it is characterised in that：The trichoderma reesei 91-3 bacterial strains are decomposed
The method of acidic cellulase is first carries out trichoderma reesei A3 after ultraviolet and nitrosoguanidine complex mutation, by treated spore
Son is inoculated on fiber double-layer plate, is cultivated 5-8 days under the conditions of 30 DEG C, is placed 7-10 days under the conditions of 15 DEG C, is selected transparent
Loop diameter and the larger single bacterium colony of colony diameter ratio carry out triangular flask solid state fermentation and screened again, obtain trichoderma reesei 91-3 bacterial strains.
8. D- trehaloses purifying technique according to claim 1, it is characterised in that：The step（8）Drying temperature is 60-
80℃。
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| CN201710221252.0A CN106947796A (en) | 2017-04-06 | 2017-04-06 | A kind of D trehaloses purifying technique |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229854A (en) * | 2019-06-24 | 2019-09-13 | 石狮市华宝明祥食品有限公司 | A kind of algae beverage processing biological enzyme processing method |
| CN117658725A (en) * | 2023-11-27 | 2024-03-08 | 山东思科生物科技有限公司 | Seaweed compound water-soluble fertilizer and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014199948A1 (en) * | 2013-06-11 | 2014-12-18 | 国立大学法人新潟大学 | Production method for β-mannoside |
| CN105586377A (en) * | 2016-01-29 | 2016-05-18 | 山东和田旺生物科技有限公司 | Enzymolysis treatment method of algae |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014199948A1 (en) * | 2013-06-11 | 2014-12-18 | 国立大学法人新潟大学 | Production method for β-mannoside |
| CN105586377A (en) * | 2016-01-29 | 2016-05-18 | 山东和田旺生物科技有限公司 | Enzymolysis treatment method of algae |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110229854A (en) * | 2019-06-24 | 2019-09-13 | 石狮市华宝明祥食品有限公司 | A kind of algae beverage processing biological enzyme processing method |
| CN117658725A (en) * | 2023-11-27 | 2024-03-08 | 山东思科生物科技有限公司 | Seaweed compound water-soluble fertilizer and preparation method and application thereof |
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