CN102323261B - Method for measuring starch content of oceanic microalgae cell - Google Patents
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Abstract
The invention discloses a method for measuring starch content of an oceanic microalgae cell. In allusion to features of the substance composition of the oceanic microalgae cell, the method comprises the following steps of: firstly, degreasing, decolorizing and desugaring the microalgae cell by using ethanol, petroleum ether and 90% ethanol; secondly, taking a calcium nitrate solution with the concentration of 0.6-0.8g/ml as an extractant for extracting starch in the microalgae cell, and boiling for 15 to 30 minutes to sufficiently extract the starch in the microalgae cell; thirdly, oxidizing residual reducing sugar and residual pigment by using hydrogen peroxide, and boiling to remove the excess hydrogen peroxide; and finally, taking iodine as a color-developing agent, and directly measuring the starch content of the oceanic microalgae through spectrophotometry. Compared with the conventional method for measuring starch content of terrestrial plants, the method has the advantages of simple process, short experimental period, accurate result and good repeatability.
Description
Technical field
The invention belongs to the sea life energy field, be specifically related to the assay method of content of starch in a kind of marine microalgae cell.
Background technology
High, individual little, nutritious, the growth and breeding of the photosynthetic utilization ratio of little algae rapidly, strong to the adaptive faculty of environment, cultivate, do not take agricultural easily and plough; Many countries have successively carried out little algae cultivation, nutritive value analysis and development and use research, have obtained many achievements and progress.Particularly in recent years, utilized marine microalgae to carry out the exploitation of biomass energy and the attention that application has attracted many researchers.Starch in the microalgae cell can be through degraded, fermentation; Finally being converted into can be by the energy that directly utilizes; A major issue that at first need solve this is how to screen little algae of being rich in starch and how to improve content of starch through optimizing culturing microalgae, and wherein the accurate mensuration of content of starch then is crucial.At present, the assay method about content of starch in the marine microalgae cell does not appear in the newspapers.The assay method of content of starch mainly reduces direct development process of iodine and reducing sugar indirect determination method two big classes in the existing terrestrial plant.The former mainly adopts water as the starch extraction agent, and starch extracts not thorough, and interfering material is difficult to remove; The latter is the method for the express-analysis mensuration starch of the J.Holm of Swede proposition in the world
[1], method is accurate, but sample needs successively to handle through AMS, amyloglucosidase, glucose oxidase in the experimentation, and three kinds of domestic being difficult to of enzyme buy simultaneously, and cost an arm and a leg.Domestic anthrone colourimetry and the enzyme hydrolysis method (GB 5009.9-85) of adopting more; All be starch in the plant to be extracted to be converted into reducing sugar earlier; Again through quantitative measurement content of reducing sugar indirect calculation content of starch; Influence accuracy as a result because intrinsic reducing sugar own is difficult for eliminating in the plant, experimental period is long, program is numerous and diverse, and these unfavorable factors are brought inconvenience to the lab analysis operation.
Summary of the invention
The object of the invention promptly is to provide a kind of and is used for sensitivity, realizes that starch in little gonidium extracts and the technical scheme of assay quickly and accurately.
The assay method of content of starch comprises following concrete steps in the marine microalgae cell of the present invention:
A. will treat that micrometer algae sample obtains little algae algae mud at the centrifugal 3～8min of 3000～3500rpm, clean repeatedly 3 times, to reach the purpose that changes the osmotic pressure inside and outside the cell when removing reaction system impurity with deionized water;
B. with behind little algae algae mud multigelation of step a gained 4～6 times, sonicated 10～20min makes microalgae cell fully broken, accurately weighs simultaneously and recording quality;
C. the little algae algae mud with step b gained mixes with absolute ethyl alcohol, behind the vibration 3min, and the centrifugal 3～8min of 3000～3500rpm, collecting precipitation; The ratio of little algae algae mud is 6ml: 1g among said absolute ethyl alcohol and the step b;
D. in step c gained deposition, add boiling range and be 60～90 ℃ sherwood oil, mix vibration 5min after, continue to add 90% ethanol, mix vibration 5min again, at centrifugal 3～8min of 3000～3500rpm and collecting precipitation; The ratio of little algae algae mud is 4ml: 1g among said sherwood oil and the step b, and the ratio of little algae algae mud is 6ml: 1g among said ethanol and the step b;
E. repeating step d is three times;
F. the calcium nitrate solution that in step e gained deposition, adds 0.6～0.8g/ml, boil 10～30min after, the starch with in the abundant extraction microalgae cell is cooled to room temperature; The ratio of little algae algae mud is 10ml: 1g among described calcium nitrate solution and the step b;
G. in step f gained solution, add 30% hydrogen peroxide, behind the oscillating reactions 20min, add 0.05mol/L hydrochloric acid, be cooled to room temperature after boiling 20min; To eliminate reducing substances and decolouring such as residual sugar and to drive residual hydrogen dioxide out of.The ratio of little algae algae mud is 2ml among the volume of the volume of said 30% hydrogen peroxide, 0.05mol/L hydrochloric acid and the step b: 1g;
H. step g gained solution is collected supernatant through the centrifugal 3～8min of 3000～3500rpm, and deposition merges all supernatants as testing sample solution again with f step re-treatment 3～5 times;
I. the testing sample solution of step h gained is with I
2-KI standard solution carries out chromogenic reaction, measures its absorbance down in the 570nm wavelength, utilizes the starch typical curve to calculate the content of starch in the testing sample solution.
For the assay method of content of starch in the above-mentioned marine microalgae cell, wherein:
The concentration that adds calcium nitrate solution among the described step f, preferably 0.8g/ml;
The number of times of re-treatment among the described step h, preferably 3 times.
Technique scheme of the present invention is to the material compositing characteristic of marine microalgae cell; At first adopting absolute ethyl alcohol, sherwood oil, 90% ethanol that microalgae cell is carried out degreasing, decolouring and desugar successively handles; And then adopt the extraction agent of the calcium nitrate solution of 0.6～0.8g/ml as starch in the microalgae cell; Boil 15～30min with the starch in the abundant extraction microalgae cell; Adopt the remaining reducing sugar of hydrogen peroxide oxidation again and eliminate residual pigment and boil and drive excessive hydrogen peroxide out of, at last with iodine as developer, utilize AAS directly to measure the content of starch in the marine microalgae; Its light absorption value is 1.3～1.4 times when directly being extraction agent with water, and the result has better accuracy.
In the said method of the present invention, the method for drafting of starch typical curve is a prior art known in the field.Those skilled in the art can according to obtainable condition confirm the scheme of optimal preparation standard curve.No matter, must strictly under equal conditions detect testing sample and calculate its content of starch with which kind of method preparation standard curve.In the embodiment of the present invention; The applicant adopts following method to prepare the starch typical curve: the 0.1g/L starch standard solution of getting different volumes; Be supplemented to equal volume with calcium nitrate solution respectively, then with the 0.01mol/L I of equal volume with step f same concentrations
2-KI standard solution colour developing back is measured its absorbance respectively under the 570nm wavelength, at last with absorbance to taken starch standard solution volume map typical curve.
This method is to the material compositing characteristic of marine microalgae cell, after marine microalgae is carried out simple pre-service, with iodine as developer; Utilize AAS directly to measure the content of starch in the marine microalgae, set up the method for content of starch in the direct quantitative mensuration marine microalgae cell, compare with the assay method of content of starch in the terrestrial plant of using always; Process is simple, and experimental period is short, and the color stability time is long; The result is accurate, favorable reproducibility.
Description of drawings
Accompanying drawing 1 width of cloth of the present invention, the typical curve that promptly prepares in the embodiment of the invention.
Embodiment
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
Experimental technique described in the following embodiment like no specified otherwise, is conventional method; Said reagent and material like no specified otherwise, all can obtain from commercial sources, or can the conventional method preparation.
Soluble starch, analyze pure, available from Chemical Reagent Co., Ltd., Sinopharm Group;
The marine microalgae that the present invention relates to all separates from the marine site, Bohai Sea Gulf.
Embodiment 1
(1) drafting of starch typical curve
Accurately take by weighing the pure starch of analysis about 0.10g, be dissolved in the deionized water that boils, be settled to the 1L volumetric flask after the cooling, be prepared into the starch standard solution; Measure this starch standard solution of different volumes again, be supplemented to 4ml with the 0.8g/mL calcium nitrate solution respectively, add 0.15mL 0.01mol/L I
2-KI standard solution shakes up, and utilizes T6 new century ultraviolet-visible pectrophotometer under the 570nm wavelength, to measure its absorbance respectively, adds 0.15mL 0.01mol/L I with the 0.8g/mL calcium nitrate solution of 4ml
2-KI standard solution is as blank, and the result sees the following form 1, to the mapping of starch standard solution volume, sees accompanying drawing 1 with absorbance.
The absorbance of table 1 different volumes standard starch solution
As shown in Figure 1; Horizontal ordinate is for being the volume of the starch standard solution of unit with ml; Ordinate A is the absorbance that utilizes T6 new century ultraviolet-visible pectrophotometer to measure down in the 570nm wavelength, and the typical curve of gained content of starch is that a related coefficient is 0.9997, slope is 0.3606 straight line.
(2) mensuration of little algae sample content of starch
A. with micro algae culturing liquid centrifugal 8min under 3000rpm, get fresh little algae algae mud;
B. fresh little algae algae mud is cleaned 3 times with 1 times deionized water respectively repeatedly, change the inside and outside osmotic pressure of cell when removing reaction system impurity;
C. with sonicated 15min behind the little algae algae mud multigelation after the washed with de-ionized water 5 times, broken microalgae cell;
D. take by weighing the little algae algae mud after 0.5191g, 0.5577g, the 0.4927g fragmentation respectively, mix with the 3ml absolute ethyl alcohol, behind the vibration 3min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.
E. in deposition, add sherwood oil 2ml and mix back vibration 5min, add the ethanol of 3ml 90% again, mix vibration 5min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.Precipitate 3 times with sherwood oil and 90% ethanol repeated washing.
F. in E gained deposition, add the calcium nitrate solution of 5ml 0.6g/mL, boil 20min and fully extract the starch in the microalgae cell, cooling solution is to room temperature.
G. in the liquid coolant of F, add the hydrogen peroxide of 1ml 30%, oscillating reactions 20min adds 1ml0.05mol/L hydrochloric acid again, is cooled to room temperature after boiling 20min, eliminates reducing substances and decolouring such as residual sugar and drives residual hydrogen dioxide out of.Centrifugal 5min under 3000rpm transfers to supernatant in the 25ml volumetric flask, and deposition merges all supernatants and is transferred in the above-mentioned 25ml volumetric flask, with 0.6g/mL calcium nitrate solution constant volume again with F step re-treatment three times.
H. from volumetric flask, shift out the 6ml sample solution in centrifugal 5min under 6000rpm.Accurately pipette the 4mL supernatant, add 0.15mL 0.01mol/L I
2-KI standard solution adds 0.15mL 0.01mol/L I with the 0.6g/mL calcium nitrate solution of 4ml
2-KI standard solution is as blank, and under the 570nm wavelength, surveying absorbance respectively is 0.494,0.572,0.476.According to absorbance, on the starch typical curve, find corresponding starch bulking value, the substitution computing formula:
Wherein:
X---the sample starch volume (ml) that checks on the typical curve
C---starch concentration of standard solution (0.1g/L)
4---the volume (ml) of taking sample determination
V---population of samples amasss (ml)
W---sample quality (g)
Calculate the sample content of starch:
Be respectively 0.161%, 0.170%, 0.163%, mean value is 0.165%.
With reference to people such as Zhang Junsong
[2]The method of introducing adopts water as the starch extraction agent, extracts the starch in this marine microalgae, and the gained result is following:
0.123%, 0.131%, 0.125%, mean value is 0.126%.
This presentation of results calcium nitrate is made the intracellular starch extraction agent of marine microalgae than the better effects if of making extraction agent with water.
Embodiment 2
The mensuration of little algae sample content of starch
A. will be cultured to marine microalgae nutrient solution centrifugal 7min under 3200rpm of balance period with seawater, get fresh little algae algae mud;
B. fresh little algae algae mud is cleaned 3 times with 1 times deionized water respectively repeatedly, change the inside and outside osmotic pressure of cell when removing reaction system impurity;
C. with sonicated 15min behind the little algae algae mud multigelation after the washed with de-ionized water 5 times, broken microalgae cell;
D. take by weighing the little algae algae mud after 0.5231g, 0.5179g, the 0.5082g fragmentation respectively, mix with the 3ml absolute ethyl alcohol, behind the vibration 3min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.
E. in deposition, add sherwood oil 2ml and mix back vibration 5min, add the ethanol of 3ml 90% again, mix vibration 5min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.Precipitate 3 times with sherwood oil and 90% ethanol repeated washing.
F. in E gained deposition, add the 0.7g/mL calcium nitrate solution of 5ml, boil 20min and fully extract the starch in the microalgae cell, cooling solution is to room temperature.
G. in the liquid coolant of F, add the hydrogen peroxide of 1ml 30%, oscillating reactions 20min adds 1ml0.05mol/L hydrochloric acid again, is cooled to room temperature after boiling 20min.Centrifugal 5min under 3000rpm transfers to supernatant in the 25ml volumetric flask, and deposition merges all supernatants and is transferred in the above-mentioned 25ml volumetric flask, with 0.7g/mL calcium nitrate solution constant volume again with F step re-treatment three times.
H. from volumetric flask, shift out the 6ml sample solution in centrifugal 5min under 6000rpm.Accurately pipette the 4mL supernatant, add 0.15mL 0.01mol/LI
2-KI standard solution adds 0.15mL 0.01mol/L I with the 0.7g/mL calcium nitrate solution of 4ml
2-KI standard solution is as blank, and under the 570nm wavelength, surveying absorbance respectively is 0.514,0.513,0.453.According to absorbance, on the starch typical curve of drawing by embodiment 1, find corresponding starch bulking value, the computing formula that substitution embodiment 1 is listed:
Calculate the sample content of starch:
Be respectively 0.166%, 0.167%, 0.161%, mean value is 0.165%.
Embodiment 3
The mensuration of little algae sample content of starch
A. will be cultured to marine microalgae nutrient solution centrifugal 5min under 3500rpm of balance period with seawater, get fresh little algae algae mud;
B. fresh little algae algae mud is cleaned 3 times with 1 times deionized water respectively repeatedly, change the inside and outside osmotic pressure of cell when removing reaction system impurity;
C. with sonicated 15min behind the little algae algae mud multigelation after the washed with de-ionized water 5 times, broken microalgae cell;
D. take by weighing little algae algae mud of getting after 0.5033g, 0.5264g, the 0.5096g fragmentation respectively, mix with the 3ml absolute ethyl alcohol, behind the vibration 3min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.
E. in deposition, add sherwood oil 2ml and mix back vibration 5min, add the ethanol of 3ml 90% again, mix vibration 5min, centrifugal 5min abandons supernatant under 3000rpm, keeps deposition.Precipitate 3 times with sherwood oil and 90% ethanol repeated washing.
F. in E gained deposition, add the 0.8g/mL calcium nitrate solution of 5ml, boil 20min and fully extract the starch in the microalgae cell, cooling solution is to room temperature.
G. in the liquid coolant of F, add the hydrogen peroxide of 1ml 30%, oscillating reactions 20min adds 1ml0.05mol/L hydrochloric acid again, is cooled to room temperature after boiling 20min.Centrifugal 5min under 3000rpm transfers to supernatant in the 25ml volumetric flask, and deposition merges all supernatants and is transferred in the above-mentioned 25ml volumetric flask, with the calcium nitrate solution constant volume of 0.8g/mL again with F step re-treatment three times.
H. from volumetric flask, shift out the 6ml sample solution in centrifugal 5min under 6000rpm.Accurately pipette the 4mL supernatant, add 0.15mL 0.01mol/L I
2-KI standard solution adds 0.15mL 0.01mol/L I with the 0.8g/mL calcium nitrate solution of 4ml
2-KI standard solution is as blank, and under the 570nm wavelength, surveying absorbance respectively is 0.507,0.506,0.514.According to absorbance, on the starch typical curve of drawing by embodiment 1, find corresponding starch bulking value, the computing formula that substitution embodiment 1 is listed:
Calculate the sample content of starch:
Be respectively 0.168%, 0.161%, 0.173%, mean value is 0.167%.
List of references:
[1]J?Holm，I?Bj?orck，A?Drews，et?al.A?rapid?method?for?the?analysis?of?starch[J].starch/starke，1986，38：224-226.
[2] Zhang Junsong, Jia Chunxiao, Mao Duobin, etc. the content of starch [J] in the iodine determination of color tobacco. tobacco science and technology, 2004, (5): 24-28.
Claims (3)
1. the assay method of the interior content of starch of marine microalgae cell is characterized in that comprising the steps:
A. will treat that micrometer algae sample obtains little algae algae mud at the centrifugal 3～8min of 3000～3500rpm, cleans 3 times with deionized water repeatedly;
B. with behind little algae algae mud multigelation of step a gained 4～6 times, sonicated 10～20min accurately weighs and recording quality;
C. the little algae algae mud with step b gained mixes with absolute ethyl alcohol, behind the vibration 3min, and the centrifugal 3～8min of 3000～3500rpm, collecting precipitation; The ratio of little algae algae mud is 6ml:1g among said absolute ethyl alcohol and the step b;
D. in step c gained deposition, add boiling range and be 60～90 ℃ sherwood oil, mix vibration 5min after, continue to add 90% ethanol, mix vibration 5min again, at centrifugal 3～8min of 3000～3500rpm and collecting precipitation; The ratio of little algae algae mud is 4ml:1g among said sherwood oil and the step b, and the ratio of little algae algae mud is 6ml:1g among said ethanol and the step b;
E. with the steps d triplicate;
F. the calcium nitrate solution that in step e gained deposition, adds 0.6～0.8g/ml is cooled to room temperature after boiling 10～30min; The ratio of little algae algae mud is 10ml:1g among described calcium nitrate solution and the step b;
G. in step f gained solution, add 30% hydrogen peroxide, behind the oscillating reactions 20min, add 0.05mol/L hydrochloric acid, be cooled to room temperature after boiling 20min; The ratio of little algae algae mud is 2ml:1g among the volume of the volume of said 30% hydrogen peroxide, 0.05mol/L hydrochloric acid and the step b;
H. step g gained solution is collected supernatant through the centrifugal 3～8min of 3000～3500rpm, and deposition merges all supernatants as testing sample solution again with f step re-treatment 3～5 times;
I. the testing sample solution of step h gained is with I
2-KI standard solution carries out chromogenic reaction, measures its absorbance down in the 570nm wavelength, utilizes the starch typical curve to calculate the content of starch in the testing sample solution:
According to absorbance, on the starch typical curve, find corresponding starch bulking value, the substitution computing formula:
Wherein:
X---the sample starch volume (ml) that checks on the typical curve
C---starch concentration of standard solution (0.1g/L)
4---the volume (ml) of taking sample determination
V---population of samples amasss (ml)
W---sample quality (g)
Calculate the sample content of starch.
2. the assay method of content of starch in a kind of marine microalgae cell according to claim 1 is characterized in that the concentration of adding calcium nitrate solution among the described step f is 0.8g/ml.
3. the assay method of content of starch in a kind of marine microalgae cell according to claim 1 and 2, the number of times that it is characterized in that re-treatment among the described step h is 3 times.
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| CN103852440A (en) * | 2012-12-05 | 2014-06-11 | 中国科学院大连化学物理研究所 | Method for measuring microalgae biomass components through Fourier transform infrared spectroscopy (FTIR) |
| CN103439279B (en) * | 2013-09-10 | 2015-04-22 | 四川农业大学 | Spectrophotometry quantitative analysis method of iodine-starch color-developing system |
| CN103645184B (en) * | 2013-12-13 | 2015-09-30 | 泸州品创科技有限公司 | The detection method of age of starch degree in a kind of vinasse |
| CN111337443B (en) * | 2020-03-30 | 2022-11-01 | 西安建筑科技大学 | Method for measuring microalgae biomass composition |
| CN112179867B (en) * | 2020-09-21 | 2022-11-29 | 上海理工大学 | Method for measuring grease in microalgae cells based on terahertz spectrum technology |
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