CN108752499A - A method of separating-purifying can obtain right polysaccharide from microbial fermentation solution - Google Patents
A method of separating-purifying can obtain right polysaccharide from microbial fermentation solution Download PDFInfo
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- CN108752499A CN108752499A CN201810433495.5A CN201810433495A CN108752499A CN 108752499 A CN108752499 A CN 108752499A CN 201810433495 A CN201810433495 A CN 201810433495A CN 108752499 A CN108752499 A CN 108752499A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
Abstract
Separating-purifying can obtain right polysaccharide from microbial fermentation solution method that the invention discloses a kind of.Production can obtain the agrobacterium zymotic fluid of right polysaccharide, and thalline were collected by centrifugation；The sediment of collection is dissolved in aqueous slkali, centrifugation removes insoluble thalline；Supernatant adds calcium chloride solution, centrifugation to obtain colloidal precipitation object；Acid supplemented solution stirs colloid and adjusts pH to 5-8, centrifugal dehydration；Colloid is washed with deionized 2-4 times, centrifugal dehydration；Gained neutral colloid is impregnated 5-10 minutes, centrifugal dehydration with 95% ethyl alcohol；Finally that gained colloid is dry, crushing obtains that right refinishing polyose product can be obtained.Method provided by the invention, aqueous slkali recyclable 70% or more, and can reuse；Simultaneously as the removal of a large amount of lye, reduces the usage amount of the acid solution for neutralization reaction, therefore, can save by a relatively large margin can obtain right separation of polysaccharides extraction cost.
Description
Technical field
The invention belongs to technical field of food industry biology, and being related to one kind separating-purifying from microbial fermentation solution can obtain so
The method of polysaccharide.
Background technology
Curdlan (Chineses：Right polysaccharide, also known as curdlan can be obtained) be one kind with carbohydrates such as glucose, sucrose
The high molecular polymer produced by microbial fermentations such as agrobacteriums (Agrobacterium sp.) for raw material.It is it is a kind of by
With β -1, straight chain polyglucose made of 3- glucosides key connections, such polydextrose has water-insoluble glucose building blocks
Property.Curdlan has the property that can quickly form gel in a heated condition, through 85 DEG C of temperatures above conditions, handles 10 points
It more than clock can form the not inverse gel of heat, therefore the thermal gels that are otherwise known as.U.S. FDA just ratified Curdlan early in 1996
As food supplementary material in food industry.Curdlan is the 3rd microorganism ratified by FDA after xanthans, gellan gum
Macromolecular glue.The Ministry of Public Health of China No. 8 bulletin approval Curdlan in 2006 is as a kind of novel food additives.GB
2760-2014 editions regulation Curdlan can be used as stabilizer, coagulator and thickener, for bean curd, Flour product, meat products, fish
In the food such as gruel.
Curdlan highly finished product, i.e., the polysaccharide after separating-purifying can just be played applied to food processing with preferable
Effect.Therefore, from zymotic fluid separating-purifying Curdlan product developments an important ring, also take up the weight of total cost of production
Want part.Currently, the rear extraction process that can obtain right polysaccharide in industrialized production is to after tunning alkaline solution treatment, use is organic
Solvent such as ethanol precipitation, dry, crushing obtain Curdlan highly finished product.The process is there are organic solvent usage amount is big, cost
Height, and there are the technological deficiencies of security risk.
Patent《A kind of separation-extraction technology of microbial polysaccharide-thermal gels》(publication number：CN1583801A one) is disclosed
The heavy Curdlan purifying techniques of kind alkali soluble acid.This method is to remove thalline by tunning alkaline solution treatment, and acid adding obtains
Gel is neutralized, then ethanol dehydration, drying, crushing obtains that right refinishing polyose product can be obtained.Patent《The rear extraction side of curdlan
Method》(publication number：CN102408490A), increase and enzymatic treatment link is carried out to zymotic fluid, while having used membrane filtration technique, but
And by the molten tunning of lye, acid solution be precipitated colloid, ethanol dehydration, drying, crushing process route obtain to obtain it is so more
Saccharin product.
The method of above-mentioned separating-purifying needs a large amount of lye, acid although reducing the dosage of ethyl alcohol in purification process
The neutralization reaction of liquid, acid, alkali raw material largely waste, of high cost；The a large amount of salt for neutralizing generation simultaneously, do not meet environmental requirement yet.
Invention content
The purpose of the present invention is for the above-mentioned of the prior art, provide it is a kind of using ionizable metal salt from microbial fermentation solution
The method that middle separating-purifying can obtain right polysaccharide.
The purpose of the present invention can be achieved through the following technical solutions：
A method of separating-purifying can obtain right polysaccharide from microbial fermentation solution, comprise the following steps that：
(1) zymotic fluid discards supernatant liquid through centrifugation or plate-frame filtering, collects thalline；
(2) under stirring conditions, the sediment that will be collected by centrifugation, according to the ratio of 1-2% (mass percentage),
It is dissolved in the sodium hydroxide or potassium hydroxide aqueous slkali of 0.2-0.5M, centrifugation or plate-frame filtering remove thalline；
(3) supernatant adds the calcium salt or magnesium salt solution of 0.1%-0.5% (mass percentage), centrifugation or squeezing to obtain glue
Body sediment；
(4) into colloidal precipitation object be added 2-4 times of quality of colloid deionized water, acid supplemented solution stir colloid adjust pH to
5-8, centrifugation or press dewatering；
(5) colloid is washed with deionized 2-4 times, centrifugation or press dewatering；
(6) gained neutral colloid is impregnated 5-10 minutes with 95% ethyl alcohol of 2-4 times of quality of colloid, centrifugation or squeezing removal
Ethyl alcohol；
(7) gained colloid is dry, crushing obtains that right refinishing polyose product can be obtained.
The preferred production of the microbial fermentation solution can obtain the agrobacterium zymotic fluid of right polysaccharide.
The condition of step (1) centrifugation removal supernatant is preferred：Rotating speed 5000-10000rmp is centrifuged 10-30 minutes.
The preferred sodium hydroxide of added aqueous slkali or potassium hydroxide solution, a concentration of 0.2-0.5M in step (2) zymotic fluid.
Calcium salt preferably calcium chloride described in step (3), the magnesium salts are magnesium chloride；The addition of the calcium salt or magnesium salts
Amount is the 0.1wt%-0.5wt% of solution.
Any one in the preferred hydrochloric acid of acid solution, sulfuric acid, acetic acid, phosphoric acid, citric acid described in step (4).
Centrifugal condition is in step (1), (2), (3), (4), (5), (6)：Rotating speed 5000-10000rmp, 10-30 points of centrifugation
Clock.
Drying means described in step (7) is in natural drying, vacuum drying, heated-air drying, ultrasonic hot-air seasoning
Any one.
The temperature of the method for the present invention all operations step is no more than 55 DEG C.
Advantageous effect：
Right refinishing polyose product can be obtained, be zymotic fluid removal thalline after obtain obtain right polysaccharide sterling, can directly apply
In food-processing industries such as plain abalone, jelly, Flour product, meat products.It is original to obtain right separation of polysaccharides method there are different to ask
Topic：Zymotic fluid alkaline solution treatment, then the method that can obtain right polysaccharide is purified through ethanol precipitation, ethanol consumption is big, of high cost；And with acid
Zymotic fluid lye is neutralized, the usage amount of the method for regathering colloid, soda acid principle is big, of high cost, while a large amount of salt generated
Environmental requirement is not met.
The present invention is different from the method that the existing separating-purifying from zymotic fluid can obtain right polysaccharide.The zymotic fluid of base extraction
It is and to form reversible curdlan body by adding calcium ion or magnesium ion salt into zymotic fluid lye after removing thalline.It collects
The lye that removal is centrifuged during colloid can reach 70% or more, this part lye may be reused.In the colloid of collection, only
Containing 30% or so lye, the use of acid solution is substantially reduced.Therefore, right separation of polysaccharides purifying technique is obtained in the present invention,
Both it solved in ethanol precipitation, the big problem of ethyl alcohol usage amount, and effectively reduces the use of acid, alkali raw material, produced simultaneously
Raw salt is also reduced therewith, meets environmental requirement.
Description of the drawings
Fig. 1 can obtain right separation of polysaccharides purifying technique schematic diagram
Specific implementation mode
The invention will be further elaborated by the following examples, but is not limitation of the invention, any according to this
The replacement of equivalent technical elements made by invention content, all without departing from protection scope of the present invention.It is according to the present invention to obtain
Right separation of polysaccharides extraction process is not limited only to that from the purification of agrobacterium separation of fermentative broth right polysaccharide can be obtained, can be obtained so from other productions
The microbial fermentation solution of polysaccharide carries out process for separating and purifying, also within protection scope of the present invention.
The method extraction that 1 this patent of embodiment provides can obtain right polysaccharide
(1) fermentation medium：Glucose 5%, (NH)2HPO40.1%, KH2PO4 0.3%, MgSO4.7H2O 0.2%,
CaCO3(1000 mesh) 0.2%, corn steep liquor 0.2% are mass percentage above, adjust solution PH to 7.
(2) fermenting microbe is agrobacterium ATCC31749, using 50L fermentation tanks, fermentation medium liquid amount 30L, fermentation
30 DEG C of cultivation temperature, speed of agitator 180r/min cultivation cycle 96h, fermenting and producing can obtain right polysaccharide.
(3) after fermentation, zymotic fluid discards supernatant liquid to agrobacterium ATCC31749 through centrifugation or plate-frame filtering, collects
Thalline, centrifugal condition are rotating speed 10000rmp, and centrifugation time is 20 minutes.
(4) thalline of collection is added to 0.3M under agitation according to the ratio of 1.5% (mass percentage)
Sodium hydroxide aqueous slkali.
(5) aqueous slkali obtained removes insoluble thalline through centrifugation, and centrifugal condition is rotating speed 10000rmp, and centrifugation time is
20 minutes.
(6) calcium chloride is added in the supernatant obtained, and additive amount is 0.2% (mass percent) of supernatant；Centrifugation obtains
Colloidal precipitation object.Centrifugal condition is rotating speed 8000rmp, and centrifugation time is 20 minutes.
(7) deionized water of 3 times of quality is added in the colloid being collected by centrifugation, and hydrochloric acid (a concentration of 3M) solution is added to stir colloid
Adjust pH to 6, centrifugal dehydration；The condition of centrifugal dehydration is rotating speed 10000rmp, and centrifugation time is 20 minutes.
(8) centrifugation gained colloid is washed with deionized 3 times, centrifugal dehydration；Centrifugal condition is rotating speed 10000rmp, centrifugation
Time is 20 minutes.
(9) centrifugation gained neutral colloid is impregnated 10 minutes with 95% ethyl alcohol of 3 times of quality, centrifugation removal ethyl alcohol.Centrifugation
Condition is rotating speed 10000rmp, centrifugation time 20 minutes.
(10) centrifugal sediment is put into baking oven, is dried under the conditions of 55 DEG C, is put into mortar and grinds after being completely dried,
Son is sieved with 100 mesh sieve to get to right refinishing polyose product can be obtained.
(11) gel strength is measured：Right refinishing polyose product can be obtained, and with pure water to be configured to 2% (mass percentage) suspended
Liquid, after homogenizer homogeneous, 90 DEG C of water bath conditions heat 10 minutes, and 4 DEG C of coolings 5 minutes obtain irreversible gel, and use
Instrumental test gel strength.
What 2 Different Extraction Method of embodiment obtained obtains right refinishing polyose product Nature comparison
By extract in 1L zymotic fluids obtain right polysaccharide for, to using what Different Extraction Method obtained to obtain right polysaccharide essence
The consumption of sodium hydroxide in the purity/gel strength/extracted amount and extraction process of product, the consumption of hydrochloric acid, ethyl alcohol
Usage amount is compared.The results are shown in Table 1.
Note：One method of patent：Method disclosed in embodiment 1 and 3 in CN1583801A, wherein lye use 0.3M's
NaOH
Two method of patent：Method disclosed in CN102408490A specific embodiment parts
Claims (8)
1. a kind of method that the separating-purifying from microbial fermentation solution can obtain right polysaccharide, it is characterised in that walked including following technique
Suddenly：
(1) zymotic fluid discards supernatant liquid through centrifugation or plate-frame filtering, collects thalline；
(2) under agitation, the sediment that will be collected by centrifugation is dissolved in sodium hydroxide or potassium hydroxide aqueous slkali, from
The heart or plate-frame filtering remove insoluble bacterial sediment；
(3) supernatant adds calcium salt or magnesium salt solution, centrifugation or squeezing to obtain colloidal precipitation object；
(4) deionized water of 2-4 times of quality of colloid is added into colloidal precipitation object, acid supplemented solution stirs colloid and adjusts pH to 5-8,
Centrifugation or press dewatering；
(5) colloid is washed with deionized 2-4 times, centrifugation or press dewatering；
(6) gained neutral colloid is impregnated 5-10 minutes with 95% ethyl alcohol of 2-4 times of quality of colloid, centrifugation or squeezing removal ethyl alcohol；
(7) gained colloid is dry, crushing obtains that right refinishing polyose product can be obtained.
2. according to the method described in claim 1, it is characterized in that the microbial fermentation solution is the soil that production can obtain right polysaccharide
Earth bacillus fermentation liquid.
3. according to the method described in claim 1, it is characterized in that step (1), (2), (3), (4), (5), (6) centrifugation go degerming
The condition of body is：Rotating speed 5000-10000rmp is centrifuged 10-30 minutes.
4. according to the method described in claim 1, it is characterized in that institute's aqueous slkali is sodium hydroxide or hydrogen in step (2) zymotic fluid
Potassium oxide solution, a concentration of 0.2-0.5M.
5. according to the method described in claim 1, it is characterized in that the calcium salt described in step (3) is calcium chloride, the magnesium salts
For magnesium chloride；The additive amount of the calcium salt or magnesium salts is the 0.1wt%-0.5wt% of zymotic fluid.
6. according to the method described in claim 1, it is characterized in that acid solution described in step (4) be hydrochloric acid, sulfuric acid, acetic acid,
Any one in phosphoric acid, citric acid.
7. according to the method described in claim 1, it is characterized in that the drying means described in step (6) is to spontaneously dry, is true
Any one in empty drying, heated-air drying, ultrasonic hot-air seasoning.
8. according to the method described in claim 1, it is characterized in that the temperature of all operations step is no more than 55 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110183547A (en) * | 2019-06-06 | 2019-08-30 | 泰兴市东圣生物科技有限公司 | One kind can obtain the efficient method for post extraction of right polysaccharide |
| CN110987717A (en) * | 2019-12-24 | 2020-04-10 | 光明乳业股份有限公司 | Analysis method of yoghourt |
| CN111172036A (en) * | 2019-12-24 | 2020-05-19 | 光明乳业股份有限公司 | Method for separating thallus from yoghourt and application of thallus in genome DNA extraction |
| CN115521959A (en) * | 2022-10-12 | 2022-12-27 | 江苏力凡胶囊有限公司 | Extraction method of pullulan |
Citations (4)
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|---|---|---|---|---|
| JPH01137990A (en) * | 1987-11-20 | 1989-05-30 | Daiichi Togyo Kk | Polysaccharide having activity for multiplying bifidobacterium |
| US4950749A (en) * | 1989-01-06 | 1990-08-21 | The Standard Oil Company | Recovery of glucan by employing a divalent cation at an alkaline pH |
| CN1583801A (en) * | 2004-06-11 | 2005-02-23 | 江南大学 | Extracting process for microbiological polysaccharide-hot gel |
| CN105420160A (en) * | 2015-12-18 | 2016-03-23 | 东华大学 | Curdlan producing strain and application thereof |
-
2018
- 2018-05-08 CN CN201810433495.5A patent/CN108752499A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01137990A (en) * | 1987-11-20 | 1989-05-30 | Daiichi Togyo Kk | Polysaccharide having activity for multiplying bifidobacterium |
| US4950749A (en) * | 1989-01-06 | 1990-08-21 | The Standard Oil Company | Recovery of glucan by employing a divalent cation at an alkaline pH |
| CN1583801A (en) * | 2004-06-11 | 2005-02-23 | 江南大学 | Extracting process for microbiological polysaccharide-hot gel |
| CN105420160A (en) * | 2015-12-18 | 2016-03-23 | 东华大学 | Curdlan producing strain and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 金阳: ""可德胶（Curdlan）多糖分子构象及其衍生物的研究"", 《中国优秀博硕士学位论文全文数据库（博士） 工程科技I辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110183547A (en) * | 2019-06-06 | 2019-08-30 | 泰兴市东圣生物科技有限公司 | One kind can obtain the efficient method for post extraction of right polysaccharide |
| CN110987717A (en) * | 2019-12-24 | 2020-04-10 | 光明乳业股份有限公司 | Analysis method of yoghourt |
| CN111172036A (en) * | 2019-12-24 | 2020-05-19 | 光明乳业股份有限公司 | Method for separating thallus from yoghourt and application of thallus in genome DNA extraction |
| CN115521959A (en) * | 2022-10-12 | 2022-12-27 | 江苏力凡胶囊有限公司 | Extraction method of pullulan |
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Application publication date: 20181106 |