CN1807465A - Mycose separation and purification method - Google Patents
Mycose separation and purification method Download PDFInfo
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- CN1807465A CN1807465A CN 200610016527 CN200610016527A CN1807465A CN 1807465 A CN1807465 A CN 1807465A CN 200610016527 CN200610016527 CN 200610016527 CN 200610016527 A CN200610016527 A CN 200610016527A CN 1807465 A CN1807465 A CN 1807465A
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Abstract
The invention discloses a Process for the purification and separation of trehalose, containing enzymolysis reaction which adds saccharify enzyme in enzyme reaction liquor which is getted from the produced trehalose by enzymic conversion method and inoculating 3-10wt% Saccharomyces cerevisiae in saccharify enzyme enzymolysis liquor, fermenting and culturing in the condition of 25-30deg C and pH 5. 5-7. 5, then getting trehalose pure product after centrifuging, hyperfiltration, ion-exchange, condensation, crystallization and desiccation. Comparing with prevenient activated charcoal column chromatography and organolite process, this invention using two-stage remove mixed sugar of enzymolysis liquor, detecting the getted trehalose pure product by HPLC, its purity is as high as 98. 0%. at the same time, this invention has the advantage of high detaching and purifying efficiency, few lose of trehalose, low cost, simple technology and so on, bringing active impel on predigesting productive technology, improving production and reducing production costs of trehalose's produce by enzyme method.
Description
Technical field
The present invention relates to a kind of separation purification method of trehalose, be meant especially and utilize enzyme transforming process to produce in the trehalose process separation purification method trehalose.
Background technology
Trehalose is to pass through α by two glucose molecules, and α-1,1 bond is closed the irreducibility disaccharide that forms.Because there is non-reduced end in trehalose; make it have protection biomass cells and biologically active substance activity under unsuitable environmental conditions such as dehydration, arid, high temperature, freezing, high osmotic pressure and toxic reagent and exempt from the destructive function; therefore, receive the increasingly extensive concern of people in recent years.
At present, Preparation methods for trehalose mainly contains microorganism extraction process, fermentation method, enzyme transforming process and gene engineering research.Wherein, enzyme transforming process is with low cost because of it, technology simple, the transformation efficiency advantages of higher has broad application prospects.Particularly utilizing malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose lytic enzyme (MTHase) and trehalose synthase (Trehalose synthase) is that the substrate enzymatic conversion method is produced the main method that trehalose has now become the trehalose suitability for industrialized production with starch, starch hydrolyzates, malto-oligosaccharide, maltose etc.With starch, starch hydrolyzates, malto-oligosaccharide, maltose etc. is that material, enzyme method prepares in the trehalose process, except containing trehalose, also contains carbohydrates such as maltose, glucose in the hydrolyzed solution behind the enzymolysis.
At present, the method for separation trehalose mainly contains activated carbon column chromatography method and ion exchange chromatography from enzymolysis solution.Gac is a non-polar adsorbent, and adsorptive power is the strongest in the aqueous solution, in organic solvent adsorptive power a little less than.Gac is to the adsorptive power difference of various sugar, and the polymerization degree of sugar is big more, and gac is strong more to its absorption.Studies show that monose such as glucose, fructose, wood sugar can be eluted from activated carbon column with roughly the same speed by water; Therefore disaccharides such as sucrose, maltose, trehalose, lactose can, can be separated the disaccharides in the enzymolysis solution, monose by this method by 5% ethanol elution, but this method can not effectively be removed a large amount of maltose in the enzymolysis solution.In addition, utilize activated carbon column to separate in the assorted sugared process trehalose loss big (more than 15%), and need a large amount of organic solvents, the trehalose production cost is increased, be not suitable for need of industrial production.Another kind method then is an ion-exchange-resin process.Because glucose has C
1The aldehyde radical of position, it dissociates under alkaline condition, makes glucose electronegative and adsorbed by anionite-exchange resin thereby discharge a H+ ion.Yet the hydroxyl of trehalose molecule dissociates than the C of glucose
1The H of position
+Ionic dissociation is wanted difficulty, according to this characteristic assorted sugar such as the maltose trisaccharide in the enzymolysis solution, maltose is converted into glucose, under alkaline condition glucose is separated with trehalose then.But this method trehalose rate of recovery has only about 49.8%, is unsuitable for industrial production requirement.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, and provide a kind of cheapness, simple method that the trehalose in the trehalose production process is separated fast and effectively with assorted sugar, simplify enzyme transforming process and produce trehalose purification procedures in the trehalose process, thereby more be adapted to industrial production requirement.
Except trehalose, also contain carbohydrates such as maltose, glucose, dextrin in enzyme transforming process production enzyme reaction solution that trehalose obtained, also have impurity such as albumen, pigment, amino acid and metal ion in addition.Therefore need just can obtain the higher trehalose of purity through serial purge process.
But the present invention is not with the free aldehyde radical according to trehalose, do not have a reductibility, all highly stable to acid and heat, not by general this characteristic of enzymic hydrolysis, utilize saccharifying enzyme that assorted sugar such as the maltose in the enzyme digestion reaction liquid, malto-oligosaccharide are hydrolyzed into glucose, utilize yeast saccharomyces cerevisiae that impurity conversion such as glucose, amino acid are become alcohol, CO then
2Deng small-molecule substance, and then reach trehalose and assorted sugared isolating purpose.
Therefore, the separation purification method of a kind of trehalose proposed by the invention, comprise in enzyme transforming process production enzyme reaction solution that trehalose obtained adding the yeast saccharomyces cerevisiae (Saccharomycescerevisiae) that saccharifying enzyme carries out enzyme digestion reaction and inoculate 3～10% weight in saccharifying enzyme enzymolysis reaction solution, cultivate at pH5.5～7.5 condition bottom fermentations.
Glucuroide, the international uniform classifying and numbering is EC.3.2.1.3, is also referred to as α-1,4-dextran glucose hydrolysis enzyme is commonly called as saccharifying enzyme.It can begin from α-1,4 key of non-reduced end, and hydrolyzed starch, maltose etc. generate glucose, and trehalose is not then had the hydrolysis effect.
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) can change into alcohol, CO rapidly with glucides such as glucose
2Deng small-molecule substance, trehalose then there is not specific action.
Yeast saccharomyces cerevisiae of the present invention belongs to: Mycophyta (Eumycophta) → Ascomycota (Ascomycophta) → Hemiascomycetes (Hemiascomycetes) or former ascus Gammaproteobacteria (Protoascomycetes) → Endomycetale (Endomycetales) or Saccharomycetes → Saccharomycetaceae (Saccharomycetaceae) → yeast subfamily (Saccharomycoideae) → yeast belong (Saccharomycopsis) → yeast saccharomyces cerevisiae kind (Saccharomyces Cerevisiae).
The present invention relates to a kind of separation purification method of trehalose particularly, may further comprise the steps:
A provides and utilizes enzyme transforming process production enzyme reaction solution that trehalose obtained, and with the enzyme reaction solution passivation, makes enzyme and hot insensitivity protein denaturation in the reaction solution;
Wherein said enzyme reaction solution can be to be raw material through the enzyme (enzyme reaction solution of malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose lytic enzyme (MTHase) and trehalose synthase (Trehalose synthase etc.) effect gained with starch, starch hydrolyzates, malto-oligosaccharide, maltose etc.; Described enzyme reaction solution can be taked 100 ℃ of passivation of high temperature;
Add saccharifying enzyme in b, the enzyme reaction solution in a step, add 0.14～0.28 saccharifying enzyme unit in every gram substrate, hydrolysis reaction 6～12h under 50～60 ℃, pH3.5-5.5 condition;
C, in saccharifying enzyme enzymolysis reaction solution, inoculate the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) of 3～10% weight,, cultivate under pH5.5～7.5 conditions at 25～30 ℃.
Describedly in saccharifying enzyme enzymolysis reaction solution, inoculate yeast saccharomyces cerevisiae, can under pH5.5～7.5 conditions, cultivate 6～16h, then 100 ℃ of passivation 10min termination reactions.
The separation purification method of related trehalose also comprises the steps:, ultrafiltration centrifugal through the fermented liquid of inoculating yeast saccharomyces cerevisiae and cultivation, passivation gained, ion-exchange, concentrated, crystallizing and drying.
Yeast saccharomyces cerevisiae used in the present invention (Saccharomyces cerevisiae) is by buying on the market, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC 1001, CICC1012, CICC1202 is available from (being called for short CICC) in Chinese industrial microbial strains preservation center; The address of depositary institution is: No. 32, (100027) Xiaoyun Road, Chaoyang District, Beijing City; Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC1001 is used to brew alcohol; Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC1012 is used to make grape wine; Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC1202 is used for beer brewing.
Key aspect of the present invention is the fermentation of Glucoamylase hydrolysis reaction and yeast saccharomyces cerevisiae:
1, Glucoamylase hydrolysis reaction
Except containing trehalose, also contain assorted sugar such as maltose, trisaccharide maltose and glucose in enzyme transforming process production enzyme reaction solution that trehalose obtained.Because maltose and trehalose are isomers, general method can not be effectively and rapidly with its separation, and the trehalose loss is bigger.The present invention utilizes saccharifying enzyme that assorted sugar such as maltose, malto-oligosaccharide are hydrolyzed into glucose for this reason, and then it is separated with trehalose.Enzyme digestion reaction finishes after the TLC method detects, result such as Fig. 1, among Fig. 1: 1. glucose mark product, 2. maltose mark product, 3. trehalose is marked product, and 4. sample behind the adding saccharifying enzyme 5. adds saccharifying enzyme in the trehalose, 6. adds saccharifying enzyme in the maltose.Only contain two kinds of carbohydrates of glucose and trehalose as seen from Figure 1 in the sample, illustrate that assorted sugar such as maltose, trisaccharide maltose and malto-oligosaccharide all are converted into glucose.It can also be seen that by Fig. 1 saccharifying enzyme does not act on trehalose, promptly can not cause the loss of trehalose.
2, fermentation by saccharomyces cerevisiae
Inoculate yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC1001, CICC1012 or CICC1202 in saccharifying enzyme enzymolysis reaction solution, it can be converted into alcohol, CO with the glucose in the enzyme digestion reaction liquid, amino acid etc.
2Deng small-molecule substance, trehalose then there is not effect.The present invention utilizes this method that glucose is separated fast and effectively with trehalose.Reaction result is seen Fig. 2, and among Fig. 2: 1. glucose is marked product, 2. maltose mark product, 3. trehalose mark product, 4. sample after the wine brewing ferment CICC1012 fermentation.As shown in Figure 2, after yeast saccharomyces cerevisiae CICC1012 fermentation, only contain a kind of glucide of trehalose in the enzyme digestion reaction liquid, and assorted sugar such as maltose, glucose are removed all.
Because the unique function that trehalose had makes trehalose at stabilate macromole and aspects such as microbial film and food fresh keeping wide application prospect be arranged.For example trehalose is used for the preservation of food, can be anticorrosion and keep the original color and luster of food; Trehalose is used for the drying process of enzyme antibody, microbial film etc., it can be transported at normal temperatures and preserves.In fact, trehalose is not extensively made major cause be that its market value is higher yet in a large number.This shows that new trehalose separation purification method proposed by the invention has reduced production cost, trehalose is applied being significant.
Compare with ion-exchange-resin process with activated carbon column chromatography method in the past, the present invention adopts the assorted sugar in the two-step approach removal enzymolysis solution, and the pure product of gained trehalose detect through HPLC, and purity reaches 98.0%, as shown in Figure 3.Advantages such as the present invention also has separation and purification efficient height, and trehalose loss is few, and is with low cost, and technology is simple, will to simplify Production by Enzymes trehalose production technique, improve output, reducing production costs produces positive pushing effect.
Description of drawings
Fig. 1. saccharifying enzyme enzymolysis afterreaction liquid detected result figure of the present invention;
Fig. 2. yeast saccharomyces cerevisiae CICC1012 fermentation of the present invention is figure as a result;
Fig. 3. gained trehalose of the present invention is through HPLC detected result figure.
Embodiment
A kind of separation purification method of trehalose may further comprise the steps:
A, provide enzyme transforming process to produce enzyme reaction solution that trehalose obtained and with passivation:
Enzyme reaction solution can be to be raw material through the enzyme reaction solution of enzyme (malt oligosaccharide based mycose synthetase MTSasc and malt oligosaccharide based mycose lytic enzyme MTHase and trehalose synthase Trehalose synthase etc.) effect gained with starch, starch hydrolyzates (W-Gum hydrolyzate), malto-oligosaccharide, maltose etc., after measured, the content of trehalose of enzyme reaction solution accounts for total reducing sugar amount about 60%, enzyme reaction solution in 100 ℃ of passivation 10min, is made enzyme and hot insensitivity protein denaturation in the reaction solution;
B, Glucoamylase hydrolysis reaction:
The transformation efficiency that generates enzyme according to sugared content and trehalose in the enzyme reaction solution is calculated assorted sugared content in enzyme transforming process production enzyme reaction solution that trehalose obtained, add saccharifying enzyme in the enzyme reaction solution in a step, add 0.14～0.28 saccharifying enzyme unit in every gram substrate, hydrolysis reaction 6～12h under 50～60 ℃, pH3.5-5.5 condition;
In addition, the saccharifying enzyme of different sources, its optimum hydrolysis temperature is also different with pH.Aspergillus is 55～60 ℃, pH3.5～5.0; Head mold is 50～55 ℃, pH4.5～5.5; Intending endomyces is 50 ℃, pH4.8～5.0.Generally hydrolysis under the low pH condition of comparatively high temps can be avoided varied bacteria growing, reduces the generation of coloring matter, is easy to decolouring.Therefore general preferred temperature 55-60 ℃.
The saccharifying enzyme that adopts energy technology company limited of Wuxi City section to produce in the present embodiment, this saccharifying enzyme is widely used in fermentation industries such as alcohol, monosodium glutamate, bent wine, beer, yellow rice wine, organic acid microbiotic, also can be used for decomposing oligose.This saccharifying enzyme optimum temperature is 58-60 ℃; The suitableeest Ph4.2-4.6 need use after stirring with 35 ℃ of-45 ℃ of warm water activation 30min during use.
C, fermentation by saccharomyces cerevisiae
In saccharifying enzyme enzymolysis reaction solution, inoculate yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC1001, CICC1012 or the CICC1202 of 3～10% weight, under 25～30 ℃, pH5.5～7.5 conditions, cultivate 6～16h, then 100 ℃ of passivation 10min termination reactions;
D, fermented liquid passivation are centrifugal
With the centrifugal 10min of gained fermented liquid 5000g among the c, remove impurity such as thalline, albumen;
E, ultrafiltration
Adopt joint to stay the above ultra-filtration equipment of molecular weight 10000Da, with the macromole Impurity removal in the reaction solution;
F, ion-exchange
The mixed bed that adopts D201 macroporous strong basic anionite-exchange resin, D001CC macropore strong acid cation exchange resin to form, yin and yang resin consumption volume ratio is 2: 1, with 2 times of deionized water wash-outs that amass to sample liquid, reaction solution is carried out desalination, decolouring processing;
G, concentrated
With reaction solution in 60 ℃, rotary evaporation under the 0.09MPa condition, being concentrated into the trehalose final concentration is 40-50%;
H, crystallizing and drying
In the trehalose concentrated solution, add the dehydrated alcohol of 4 times of volumes, and drop into crystal seed, 4 ℃ of static 12h, trehalose is promptly crystallizable.The trehalose crystallization is filtered,, be the pure product of trehalose in 40 ℃ of following vacuum-drying 10h then with the small amount of ethanol flushing.
Claims (8)
1, a kind of separation purification method of trehalose, comprise in enzyme transforming process production enzyme reaction solution that trehalose obtained adding the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) that saccharifying enzyme carries out enzyme digestion reaction and inoculate 3～10% weight in saccharifying enzyme enzymolysis reaction solution, cultivate at pH5.5～7.5 condition bottom fermentations.
2, the separation purification method of a kind of trehalose according to claim 1 may further comprise the steps:
A, provide and utilize enzyme transforming process to produce the enzyme reaction solution that trehalose obtained, and, make enzyme and hot insensitivity protein denaturation in the reaction solution the enzyme reaction solution passivation;
Add saccharifying enzyme in b, the enzyme reaction solution in a step, add 0.14～0.28 saccharifying enzyme unit in every gram substrate, hydrolysis reaction 6～12h under 50～60 ℃, pH3.5-5.5 condition;
C, in saccharifying enzyme enzymolysis reaction solution, inoculate the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) of 3～10% weight,, cultivate under pH5.5～7.5 conditions at 25～30 ℃.
3, ask the separation purification method of 2 described a kind of trehaloses according to right, it is characterized in that the described yeast saccharomyces cerevisiae cultivation 6～16h that in saccharifying enzyme enzymolysis reaction solution, inoculates, then 100 ℃ of passivation 10min termination reactions.
4, the separation purification method of a kind of trehalose according to claim 3 also comprises the steps:, ultrafiltration centrifugal through the fermented liquid of inoculating yeast saccharomyces cerevisiae and cultivation, passivation gained, ion-exchange, concentrated, crystallizing and drying.
5, the separation purification method of a kind of trehalose according to claim 4, it is characterized in that the mixed bed that described ion-exchange adopts D201 macroporous strong basic anionite-exchange resin, D001-CC macropore strong acid cation exchange resin to form, yin and yang resin consumption volume ratio is 2: 1, with 2 times of deionized water wash-outs that amass to sample liquid, reaction solution is carried out desalination, decolouring processing.
6, the separation purification method of a kind of trehalose according to claim 4, it is characterized in that described concentration process be with the reaction solution of decolouring after the desalination in 60 ℃, rotary evaporation under the 0.09MPa condition, being concentrated into the trehalose final concentration is 40-50%.
7, the separation purification method of a kind of trehalose according to claim 1 and 2 is characterized in that described enzyme reaction solution is is the enzyme reaction solution of raw material through enzyme effect gained with starch, starch hydrolyzates, malto-oligosaccharide, maltose etc.
8,, it is characterized in that described yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC 1001, CICC 1012 or CICC1202 according to the separation purification method of any one described a kind of trehalose in the claim 1 to 7.
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| CN101793884A (en) * | 2010-04-06 | 2010-08-04 | 山东轻工业学院 | Method for detecting content of trehalose in artemia eggs |
| CN103125905A (en) * | 2011-11-25 | 2013-06-05 | 玉米产品开发股份有限公司 | Method for preparing high purity gentiooligosaccharide, high purity gentiooligosaccharide obtained therefrom and uses thereof |
| CN103450288A (en) * | 2013-08-16 | 2013-12-18 | 齐鲁工业大学 | Separation and purification method of trehalose |
| CN104177449A (en) * | 2014-07-24 | 2014-12-03 | 菏泽学院 | Environment-friendly production method for enhancing trehalose yield of beer waste yeast under stress conditions |
| CN104592316A (en) * | 2015-01-31 | 2015-05-06 | 湖南尔康制药股份有限公司 | Preparation method for trehalose for injection |
| CN105925642A (en) * | 2016-06-14 | 2016-09-07 | 湖南汇升生物科技有限公司 | Method of industrial production for trehalose by way of microbial fermentation |
| CN106244647A (en) * | 2016-08-24 | 2016-12-21 | 山东福洋生物科技有限公司 | A kind of method simultaneously preparing trehalose and gluconic acid lactone |
| CN106317131A (en) * | 2016-08-24 | 2017-01-11 | 山东福洋生物科技有限公司 | Crystallization method of mycose |
| CN107001410A (en) * | 2014-12-08 | 2017-08-01 | Emd密理博公司 | Mixed bed ion-exchange adsorbent |
| CN111978423A (en) * | 2020-08-26 | 2020-11-24 | 保龄宝生物股份有限公司 | Preparation method of high-purity galactooligosaccharide |
| CN112079885A (en) * | 2020-09-14 | 2020-12-15 | 江苏省奥谷生物科技有限公司 | Preparation method of anhydrous trehalose |
| US11305271B2 (en) | 2010-07-30 | 2022-04-19 | Emd Millipore Corporation | Chromatography media and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101793884B (en) * | 2010-04-06 | 2012-07-18 | 山东轻工业学院 | Method for detecting content of trehalose in artemia eggs |
| CN101793884A (en) * | 2010-04-06 | 2010-08-04 | 山东轻工业学院 | Method for detecting content of trehalose in artemia eggs |
| US11305271B2 (en) | 2010-07-30 | 2022-04-19 | Emd Millipore Corporation | Chromatography media and method |
| CN103125905A (en) * | 2011-11-25 | 2013-06-05 | 玉米产品开发股份有限公司 | Method for preparing high purity gentiooligosaccharide, high purity gentiooligosaccharide obtained therefrom and uses thereof |
| CN103450288A (en) * | 2013-08-16 | 2013-12-18 | 齐鲁工业大学 | Separation and purification method of trehalose |
| CN103450288B (en) * | 2013-08-16 | 2016-05-11 | 齐鲁工业大学 | A kind of isolation and purification method of trehalose |
| CN104177449A (en) * | 2014-07-24 | 2014-12-03 | 菏泽学院 | Environment-friendly production method for enhancing trehalose yield of beer waste yeast under stress conditions |
| CN104177449B (en) * | 2014-07-24 | 2016-08-17 | 菏泽学院 | Stress conditions is utilized to improve the environment-protection production method of beer waste yeast trehalose yield |
| CN107001410A (en) * | 2014-12-08 | 2017-08-01 | Emd密理博公司 | Mixed bed ion-exchange adsorbent |
| US11236125B2 (en) | 2014-12-08 | 2022-02-01 | Emd Millipore Corporation | Mixed bed ion exchange adsorber |
| CN104592316A (en) * | 2015-01-31 | 2015-05-06 | 湖南尔康制药股份有限公司 | Preparation method for trehalose for injection |
| CN105925642A (en) * | 2016-06-14 | 2016-09-07 | 湖南汇升生物科技有限公司 | Method of industrial production for trehalose by way of microbial fermentation |
| CN105925642B (en) * | 2016-06-14 | 2019-08-13 | 湖南汇升生物科技有限公司 | With the method for microbe fermentation method industrialized production trehalose |
| CN106317131B (en) * | 2016-08-24 | 2019-04-16 | 山东福洋生物科技有限公司 | A kind of method for crystallising of trehalose |
| CN106244647B (en) * | 2016-08-24 | 2020-04-24 | 德州汇洋生物科技有限公司 | Method for simultaneously preparing trehalose and gluconolactone |
| CN106317131A (en) * | 2016-08-24 | 2017-01-11 | 山东福洋生物科技有限公司 | Crystallization method of mycose |
| CN106244647A (en) * | 2016-08-24 | 2016-12-21 | 山东福洋生物科技有限公司 | A kind of method simultaneously preparing trehalose and gluconic acid lactone |
| CN111978423A (en) * | 2020-08-26 | 2020-11-24 | 保龄宝生物股份有限公司 | Preparation method of high-purity galactooligosaccharide |
| CN112079885A (en) * | 2020-09-14 | 2020-12-15 | 江苏省奥谷生物科技有限公司 | Preparation method of anhydrous trehalose |
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