CN107132149A - A kind of method of quick specific detection curdlan content - Google Patents
A kind of method of quick specific detection curdlan content Download PDFInfo
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- CN107132149A CN107132149A CN201710235584.4A CN201710235584A CN107132149A CN 107132149 A CN107132149 A CN 107132149A CN 201710235584 A CN201710235584 A CN 201710235584A CN 107132149 A CN107132149 A CN 107132149A
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Abstract
The invention belongs to food and fermented product detection technique field, and in particular to a kind of method and its application of quick specific detection curdlan content.A kind of method of quick specific detection curdlan content, comprises the following steps：Preparation, the preparation of curdlan standard alkali solution, the making of curdlan standard curve and the curdlan sample size of aniline blue solution are determined.This method the change of curdlan yield and can accordingly adjust fermenting and producing strategy, quickly compare production capacity of the different strains during liquid fermentation, so as to promote curdlan strain improvement and ferment control level in the various solution of quick detection such as zymotic fluid in the content of curdlan, quick tracking fermentation process.
Description
Technical field
The invention belongs to food and fermented product detection technique field, and in particular to a kind of quick specific detection gel is more
The method and its application of sugared content.
Background technology
Curdlan (curdlan) is the glucan being polymerized by β -1,3-D glycosidic bonds, can utilize grape by microorganism
The carbon source through fermentation such as sugar, sucrose are produced.Curdlan is widely used in fields such as food, medicine, feed, health cares, is a kind of high attached
Value added product.
The measure for curdlan content mainly passes through phenol Sulphuric acid colorimetry, Congo red development process and extracted and claims at present
The modes such as weight method.The principle of wherein phenol Sulphuric acid Colorimetry polysaccharide is that polysaccharide first is hydrolyzed into monose such as under concentrated acid effect
Glucose is quickly generated furfural derivatives and then occurs chromogenic reaction with phenol again, and the kind of polysaccharide is cannot distinguish between in this way
Class and glucosides key type, can not distinguish polysaccharide and small molecular sugar mixture, so the detection to curdlan lacks specificity；
Congo red development process can also determine containing for many kinds of polysaccharide using the Congo red displacement that can be complexed with polysaccharide and occur absorbing wavelength
Amount, but can be more with the Congo red many sugar types for occurring chromogenic reaction, such as cellulose, chitosan, dextran (α-D-
1-6- glucans) etc., therefore Congo red development process is still without curdlan specificity；Extracting weight method needs to utilize gel many
The characteristic of sugar will contain curdlan sample and carry out acid adding precipitation, then obtained through washing for several times with alcohol precipitation again after alkali soluble solution centrifugation
Dried after pure curdlan sample to constant weight, process is cumbersome time-consuming very long.These methods are all difficult timely and accurately to analyze each
Plant the curdlan content in product, it is also difficult to timely and accurately reflect the dynamic change of curdlan yield in fermentation process.
Such as, if including variety classes polysaccharide, monose and oligosaccharides simultaneously in fluid product or zymotic fluid, without isolating and purifying use
Phenol sulfuric acid procedure and Congo red development process all can not Accurate Determining, and isolate and purify various carbohydrates row detection then expense again if desired
When it is laborious.Therefore, a kind of method for being capable of quick specific detection curdlan content is developed to have great importance.
There are some researches show β -1,3-D- glucans can be dyed exclusively by aniline blue, this is applied in solid medium
Add aniline blue and curdlan producing strains are carried out with dyeing identification, but be due to aniline blue ability only under neutral and acid condition
Stable blueness is shown, and curdlan can only dissolve in the basic conditions, this causes aniline blue decoration method for a long time
It can be only applied to primary dcreening operation of the curdlan producing strains on solid plate culture medium and be not used to the quantitative analysis of curdlan.
We have found that brownish red is presented initial stage with curdlan in aniline blue in the basic conditions under study for action, then quick to fade, and treat one
Colour generation is just gradually stablized and with accordingly stable spectral absorption after fixing time, now its OD value and the linear phase of curdlan concentration
Close.When this curdlan and reaction of the aniline blue in aqueous slkali are by concentration of substrate, reaction system, reaction temperature, reaction
Between etc. factor significantly affect, it is necessary to by the optimization of various conditions, can just draw and coagulated using the quick specific detection of aniline blue
Curdlan content in the method for glue polysaccharide content, especially quick detection liquid, for curdlan strain improvement and hair
The control of ferment production process, optimization of process conditions have extremely important meaning.
The information for being disclosed in the background section is merely intended to understanding of the increase to the general background of the present invention, without answering
When the prior art for being considered as recognizing or implying the information structure in any form well known to persons skilled in the art.
The content of the invention
It is an object of the invention to provide a kind of method of quick specific detection curdlan content, gel is more in this method
Sugared content is related in good linear to absorbance, can obtain R2>0.9 standard curve.
A kind of method of quick specific detection curdlan content, comprises the following steps：
(1) preparation of aniline blue solution：Aniline blue solid is weighed, deionized water stirring is added, held after being used after abundant dissolving
Measuring bottle constant volume, is configured to aniline blue solution；
(2) preparation of curdlan standard alkali solution：It is accurate to weigh drying to the curdlan mark product of constant weight in beaker,
Addition 60mL 0.1mol/LNaOH solution, which is stirred to polysaccharide, to be completely dissolved, then is settled to 100mL with 0.1mol/L NaOH solutions,
Obtain curdlan standard alkali solution；
(3) making of curdlan standard curve：A series of curdlan standard alkali solution of different volumes is taken respectively, plus
Enter 0.1mol/L NaOH solutions and supply 1mL, obtain a series of prepare liquid of different curdlan final concentrations, then be separately added into
100 μ L aniline blue solution are mixed therewith, are immediately placed in 25-40 DEG C of water-bath 20-40min, are then immediately placed in ice-water bath
In, draw 200 μ L reaction solutions and add in ELISA Plate, absorbance is determined with ELIASA, then using absorbance as ordinate, with gel
Polysaccharide mark product concentration is abscissa, draws standard curve；
(4) curdlan sample size is determined：Fermentation broth sample containing curdlan is added into NaOH solution, oscillator is used
Shaken well, to being completely dissolved, centrifuges 5-10min, it is curdlan sample alkali soluble to take supernatant with 6000-10000r/min
Liquid, makes the final concentration of 0.1mol/L of its NaOH with deionized water dilution curdlan sample aqueous slkali, is then surveyed by standard curve
Determine the same step of method and determine absorbance, substitute into standard curve and calculate sample size.
Preferably, the mass percent of aniline blue solution is 0.1-1% in step (1).
Preferably, the measure wavelength of ELIASA described in step (3), (4) is 540-560nm.
Preferably, the curdlan and aniline blue described in step (3), (4) are anti-in pH8-14 alkaline solution
Should.
Compared with prior art, the present invention has the advantages that：
1. the phenol Sulphuric acid colorimetry that can solve to commonly use at present using this method, Congo red colorimetric method to curdlan without
Specific shortcoming, can also solve extract weight method process it is cumbersome, take too long of deficiency.
2. this method can the content of curdlan in the various solution of quick detection such as zymotic fluid, quickly track fermentation process
The change of middle curdlan yield simultaneously accordingly adjusts fermenting and producing strategy, quickly compares different strains during liquid fermentation
Production capacity, so as to promote curdlan strain improvement and ferment control level.
3. the curdlan content in various solids, liquid, solid-liquid mixture product can also be carried out using this method
Detection, can be prepared into after aqueous slkali for the solid sample containing curdlan and determine.
Brief description of the drawings
Fig. 1 is the curdlan content standard curve that the inventive method makes using commercially available curdlan as standard items；
The curdlan content standard curve that the curdlan that Fig. 2 is the inventive method to extract from zymotic fluid makes；
Fig. 3 is that the inventive method is more to levan, soluble starch, sanlose, dextran and gel
The measurement result of sugared 5 kinds of samples compares figure；
Fig. 4 uses length scanning collection of illustrative plates for ELIASA in the inventive method.
Embodiment
The embodiment of the present invention is described in detail with reference to embodiment, it is to be understood that the guarantor of the present invention
Shield scope is not limited by embodiment.
The strain that the present invention is used can be bought from DSMZ, can also pass through field acquisition or other approach
Obtain.It is mature technology commonly used in the art that microculture and curdlan, which such as extract at the related technical scheme, is related to
Technical term is also common technical term in this area, and embodiment is illustrative, rather than limited, it is impossible to explained
To limit protection scope of the present invention.
Embodiment 1：
A kind of method of quick specific detection curdlan content, standard is made by mark product of commercially available curdlan bent
Line, comprises the following steps：
(1) preparation of aniline blue solution：It is accurate to weigh 0.5g aniline blue solids, deionized water stirring is added, treats fully molten
With volumetric flask constant volume to 100mL after solution, 0.5% water-soluble aniline blue solution is configured to；
(2) preparation of curdlan standard alkali solution：Commercially available curdlan mark product are dried to constant weight, accurately weighed
0.25g adds 60mL 0.1mol/LNaOH solution magnetic agitations to polysaccharide and is completely dissolved, then use 0.1mol/L in beaker
NaOH solution is settled to 100mL, obtains 0.25% curdlan standard alkali solution；
(3) making of curdlan standard curve：With 0.25% curdlan standard alkali solution, a series of differences are taken respectively
The curdlan standard alkali solution of volume, adds 0.1mol/L NaOH solutions and supplies 1mL, obtain curdlan final concentration of
0.05-0.10% prepare liquid, adds 100 μ L aniline blue solution, is immediately placed in 35 DEG C of water-bath 30min, then rapidly
It is placed in ice-water bath, draws 200 μ L reaction solutions and add in ELISA Plate, absorbance OD is determined with ELIASA550, with OD550For vertical seat
Mark, standard curve is drawn by abscissa of curdlan mark product concentration；
As a result as shown in figure 1, obtaining calibration curve equation for y=9.58794x-0.27211, its R2=0.99788, line
Property it is good, illustrate that the inventive method can carry out quantitative analysis.
Embodiment 2：
A kind of method of quick specific detection curdlan content, extracts curdlan sterling making mark from zymotic fluid
Directrix curve is simultaneously applied to curdlan content colorimetric estimation in zymotic fluid, comprises the following steps：
(1) prepared by curdlan zymotic fluid：With the strain of life science and technology institute of Guangxi University preservation
Exemplified by Agrobacterium radiobacter A-15, it is transferred to seed culture from inclined-plane and is based on 32 DEG C, 220r/min shaking tables
18h is cultivated, then is transferred to by inoculum concentration 12% in fermentation medium, gel is obtained in 32 DEG C, 240r/min shaking table cultures 120h
Polysaccharide fermentation liquid；
Described seed culture medium is 0.3% (NH4)2HPO4, 2% Dextrose Monohydrate, 0.15%KH2PO4, 0.1%
MgSO4, 0.1% corn steep liquor, pH7.2；
Described fermentation medium is 5% glucose, 0.03% (NH4)2HPO4, 0.2%KH2PO4, 0.05%MgSO4,
0.05% corn steep liquor, pH7.2.
(2) from broth extraction curdlan：The zymotic fluid described in 1mL the present embodiment step (1) is drawn to manage in 2mL EP
In, 1mol/L NaOH are added to 2mL, oscillator shaken well is used, and to being completely dissolved, 8000r/min centrifuges 5min.Draw 1mL
Supernatant adds 2mol/L HCl to 2mL in 2mL EP pipes, uses oscillator shaken well, and 12000r/min centrifuges 10min,
Supernatant is discarded, curdlan precipitation is obtained；
Curdlan precipitation is added into deionized water to 2mL, oscillator shaken well is used, stands 10min, then
12000r/min centrifuges 10min, discards supernatant, and precipitation adds absolute ethyl alcohol to 2mL, uses oscillator shaken well, stands
10min, 12000r/min centrifuge 10min, discard supernatant.This step 3 time is repeated, supernatant discarding retains what is extracted in EP pipes
Curdlan is precipitated；
The curdlan of extraction precipitation and EP pipes are placed in rotatory vacuum drying instrument 60 DEG C of dry 3h-5h to perseverance together
Weight, that is, obtain the solid mark product of broth extraction curdlan；
(3) preparation of aniline blue solution：It is accurate to weigh 0.5g aniline blue solids, deionized water stirring is added, treats fully molten
Volumetric flask constant volume 100mL is used after solution, 0.5% water-soluble aniline blue solution is configured to；
(4) preparation of broth extraction curdlan standard alkali solution：By obtained by the present embodiment step (2) from zymotic fluid
The curdlan solid mark product of extraction are dried to constant weight, accurately weigh 0.25g in beaker, add 60mL 0.1mol/LNaOH
Solution magnetic agitation to polysaccharide is completely dissolved, then is settled to 100mL with 0.1mol/L NaOH solutions, and it is 0.25% to obtain concentration
Broth extraction curdlan standard alkali solution；
(5) making of broth extraction curdlan standard curve：A series of different volumes the present embodiment steps are taken respectively
(4) the broth extraction curdlan standard alkali solution obtained by, adds 0.1mol/L NaOH solutions and supplies 1mL, obtain gel
The final concentration of 0.1-0.2% of polysaccharide prepare liquid, adds 100 μ L aniline blue solution, is immediately placed in 35 DEG C of water-baths
30min, is then immediately placed in ice-water bath, draws 200 μ L reaction solutions and adds in ELISA Plate, absorbance is determined with ELIASA
OD550, with absorbance OD550For ordinate, standard curve is drawn by abscissa of the concentration of broth extraction curdlan；
(6) in zymotic fluid curdlan content colorimetric estimation：1mL zymotic fluids are taken to add 1mol/L NaOH solutions extremely
2mL, uses oscillator shaken well, and to being completely dissolved, 8000r/min centrifuges 5min, and it is curdlan zymotic fluid to take supernatant
Sample aqueous slkali, makes the final concentration of 0.1mol/L of its NaOH, so with deionized water dilution curdlan fermentation broth sample aqueous slkali
Reacted afterwards by the same process of the present embodiment step (5), determine absorbance OD550, substitute into obtained by the present embodiment step (5)
Standard curve calculates the content of curdlan in fermentation broth sample.
The present embodiment acquired results are as shown in Fig. 2 obtain calibration curve equation for y=5.37361x-0.3208, its R2=
0.99738, it is linear good, illustrate that the inventive method can carry out quantitative analysis.The standard curve with reference to obtained by the present embodiment, with this
The content of curdlan in zymotic fluid obtained by inventive method aniline blue colorimetric determination the present embodiment step (1), and claim with extracting
Weight method compares, and the results are shown in Table 1.
The colorimetric method of table 1 and dry weight method determine the comparison of zymotic fluid curdlan content
As shown in table 1, the inventive method is relatively coincide with extracting the result of weight method measurement.In the present embodiment, with the present invention
Method determines the standard deviation of zymotic fluid curdlan concentration in 0.019-0.069%, is compared with extracting weight method measurement result,
Average relative error is 4.98%.
The inventive method can be used for the colorimetric estimation of zymotic fluid curdlan content, and its measurement result is with extracting weight method
It is close, but process is more simple and quick than extracting weight method.
Embodiment 3：
A kind of method of quick specific detection curdlan content, to the specific detection of curdlan, including it is following
Step：
(1) preparation of aniline blue solution：It is accurate to weigh 0.5g aniline blue solids, deionized water stirring is added, treats fully molten
With volumetric flask constant volume to 100mL after solution, 0.5% water-soluble aniline blue solution is configured to；
(2) preparation of curdlan standard alkali solution：Commercially available curdlan mark product are dried to constant weight, accurately weighed
0.25g adds 60mL 0.1mol/LNaOH solution magnetic agitations to polysaccharide and is completely dissolved, then use 0.1mol/L in beaker
NaOH solution is settled to 100mL, obtains 0.25% curdlan standard alkali solution；
(3) preparation of other types polysaccharide standard alkali solution：Respectively by commercially available soluble starch, sodium carboxymethylcellulose
Salt, the homemade levan sample of dextran mark product and Guangxi University are dried to constant weight, accurately weigh 0.25g in beaker, plus
Enter 60mL 0.1mol/LNaOH solution magnetic agitations to polysaccharide to be completely dissolved, then be settled to 0.1mol/L NaOH solutions
100mL, obtain concentration be 0.25% levan, soluble starch, sanlose, dextran standard alkali soluble
Liquid；
(4) specific detection：400 μ L concentration are taken as blank control, respectively using the 0.1mol/L NaOH solutions without sugar
Be 0.25% curdlan standard alkali solution, levan standard alkali solution, soluble starch standard alkali solution, carboxymethyl it is fine
The plain sodium salt standard alkali solution of dimension, dextran standard alkali solution, supply 1mL with 0.1mol/L NaOH solutions, obtain all kinds of
Type polysaccharide prepare liquid, adds 100 μ L aniline blue solution, is immediately placed in 35 DEG C of water-bath 30min, is then immediately placed in frozen water
In bath, draw 200 μ L reaction solutions and add in ELISA Plate, absorbance OD is determined with ELIASA550, comparative measurements result.
As a result as shown in figure 3, successively with blank and levan, soluble starch, the sodium carboxymethylcellulose of comparable sodium
Salt, dextran and curdlan mark product are made comparisons measure, are as a result shown except curdlan has obvious OD550Outside light absorption value,
The OD of the polysaccharide of remaining several frequently seen non-beta -1,3-D glycosidic bond composition550It is suitable with blank, far below gel under equal conditions
The absorption of polysaccharide, illustrates that the polysaccharide of non-beta -1,3-D glycosidic bond composition disturbs negligible to this method, the inventive method is more to gel
The specificity of sugar is preferably.
Embodiment 4：
A kind of method of quick specific detection curdlan content, the wavelength that the ELIASA is used when determining is to pass through
Curdlan is reacted in the basic conditions with aniline blue, reaction solution is subjected to absorbing wavelength scanning after terminating, absorbance is selected
Larger wavelength period is used to detect, comprised the following steps：
(1) absorbing wavelength is scanned：Using according to the curdlan standard alkali solution obtained by the step of embodiment 1 (2)
0.075% prepare liquid, adds 100 μ L aniline blue solution, is immediately placed in 30 DEG C of water-bath 20min, is then immediately placed in frozen water
In bath, draw 200 μ L reaction solutions and add in ELISA Plate, absorbing wavelength scanning is carried out in 450-600nm scopes with ELIASA.
As a result as shown in figure 4, explanation the inventive method is larger in wavelength 540-560nm scope internal absorbances, and OD values exist
In the range of 0.2-0.8, therefore the wavelength period can be used for the inventive method colorimetric estimation.
It is foregoing to the present invention specific illustrative embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, can be much changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that the certain principles and its reality of the present invention should
With so that those skilled in the art can realize and using the present invention a variety of exemplaries and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. a kind of method of quick specific detection curdlan content, it is characterised in that comprise the following steps：
(1) preparation of aniline blue solution：Aniline blue solid is weighed, deionized water stirring is added, volumetric flask is used after fully dissolving
Constant volume, is configured to aniline blue solution；
(2) preparation of curdlan standard alkali solution：It is accurate to weigh drying to the curdlan mark product of constant weight in beaker, addition
60mL 0.1mol/L NaOH solutions, which are stirred to polysaccharide, to be completely dissolved, then is settled to 100mL with 0.1mol/L NaOH solutions, is obtained
To curdlan standard alkali solution；
(3) making of curdlan standard curve：A series of curdlan standard alkali solution of different volumes is taken respectively, is added
0.1mol/L NaOH solutions supply 1mL, obtain a series of prepare liquid of different curdlan final concentrations, then are separately added into 100
μ L aniline blue solution is mixed therewith, is immediately placed in 25-40 DEG C of water-bath 20-40min, is then immediately placed in ice-water bath,
Draw 200 μ L reaction solutions to add in ELISA Plate, absorbance is determined with ELIASA, it is many with gel then using absorbance as ordinate
Sugared mark product concentration is abscissa, draws standard curve；
(4) curdlan sample size is determined：Fermentation broth sample containing curdlan is added into NaOH solution, vibrated with oscillator
Uniformly, to being completely dissolved, 5-10min is centrifuged with 6000-10000r/min, it is curdlan sample aqueous slkali to take supernatant,
Make the final concentration of 0.1mol/L of its NaOH with deionized water dilution curdlan sample aqueous slkali, then by standard curve determination side
The same step of method determines absorbance, substitutes into standard curve and calculates sample size.
2. the method for quick specific detection curdlan content according to claim 1, it is characterised in that step (1)
The mass percent of middle aniline blue solution is 0.1-1%.
3. the method for quick specific detection curdlan content according to claim 1, it is characterised in that step (3),
(4) the measure wavelength of ELIASA described in is 540-560nm.
4. the method for quick specific detection curdlan content according to claim 1, it is characterised in that step (3),
(4) curdlan and aniline blue described in are the reactions in pH8-14 alkaline solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760708A (en) * | 2018-07-04 | 2018-11-06 | 无限极（中国）有限公司 | A kind of rapid detection method of triple-helix structure lentinan |
| CN109115907A (en) * | 2018-08-21 | 2019-01-01 | 广西大学 | A kind of levan molecular weight determination and the method for separation preparation |
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