CN104950006A - Method for rapidly determining content of polyunsaturated fatty acids in oil-containing microorganism cells - Google Patents
Method for rapidly determining content of polyunsaturated fatty acids in oil-containing microorganism cells Download PDFInfo
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- CN104950006A CN104950006A CN201410118153.6A CN201410118153A CN104950006A CN 104950006 A CN104950006 A CN 104950006A CN 201410118153 A CN201410118153 A CN 201410118153A CN 104950006 A CN104950006 A CN 104950006A
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Abstract
The invention discloses a method for rapidly determining the content of polyunsaturated fatty acids in oil-containing microorganism cells. The method is used for mainly detecting the relative content of polyunsaturated fatty acids in the cells in microorganisms including microalgae, bacteria, fungi, yeast and the like; after a cell culture solution is centrifuged, the bacteria are re-suspended in a buffering solution, and a nuclear magnetic resonance 1H spectrum or 13C spectrum is determined; and the contents of polyunsaturated fatty acids of the different samples can be compared according to the strength ratio of a characteristic peak on a nuclear magnetic resonance spectrogram. Compared with a traditional fatty acid content determination method, operations including sample drying, cell crushing, grease extraction, transesterification and the like are not needed and an organic solvent is not needed, so that manpower and material resources are saved and the determination efficiency is greatly improved; and the method is suitable for high-throughput rapid detection of a lot of samples, such as microorganism screening, optimization of culture conditions and monitoring of an industrial production process.
Description
Technical field
The invention belongs to detection method technical field, be specifically related to the method for content of polyunsaturated fatty acid in a kind of cell, particularly relate to the method for unsaturated fatty acid content in a kind of quick judgement cell being applicable to the monitoring of bacterial screening, microorganism training systern and commercial production polyunsaturated fatty acid microbial cultivation process.
Background technology
Polyunsaturated fatty acid (mainly comprising DHA, EPA, ARA etc.) has and maintains Cell Homeostasis and normal growth, angiocardiopathy preventing, immunological regulation and many important physiological actions such as anticancer.Polyunsaturated fatty acid is long-term to be used as the important additives in baby milk powder, health food, medicine, also starts in recent years to improve its nutritive value as adjuvant in the food such as edible oil, dairy produce.Early stage polyunsaturated fatty acid is mainly derived from deep sea fish oil, and the impact by factors such as overfishing, environmental pollution, seasonal variations is very large.Microbe-derived polyunsaturated fatty acid then has impact with short production cycle, not to be subject to season and place, not by features such as physical environment pollution effects, become the main production source of the polyunsaturated fatty acid of alternative deep sea fish oil gradually, and be widely used in scientific research and suitability for industrialized production.The microorganism producing polyunsaturated fatty acid comprises micro-algae, bacterium, fungi, yeast etc., and these microorganisms are usually to produce a certain polyunsaturated fatty acid; High yield DHA as usual in schizochytrium, Shewanella high yield EPA, Mortierella alpina high yield ARA etc.
The microorganism producing polyunsaturated fatty acid usually separation and purification from physical environment (bacterial strain screening) obtains, and carries out medium optimization, training systern and amplification cultivation etc., finally for large-scale industrial production by methods such as Fermentation Engineerings.In bacterial strain screening, training systern, commercial process, need to measure the content of polyunsaturated fatty acid in microorganism, to reach the object improving output or monitor production process.The method of current mensuration fatty acid composition needs first to extract grease usually, then uses the methods such as GC, GC-MS, HPLC or NMR to measure composition and the content of fatty acid in grease.Sample required for these methods needs the step by solvent extraction and derivatization etc. are complicated to be prepared usually, not only length consuming time, and may have an impact to accuracy of detection, causes larger error.Therefore, find the method for simple and quick mensuration unsaturated fatty acid, batch samples in bacterial strain screening, training systern, commercial process is detected and has great importance.
Summary of the invention
The object of the present invention is to provide the method for content of polyunsaturated fatty acid in a kind of quick detection microbial cell.The method utilizes microorganism self can high Lipid-producing form fat and drip this characteristic, direct use cell carries out magnetic resonance detection, by the strength ratio of the characteristic peak of saturated fatty acid on nmr spectrum and unsaturated fatty acid, judge polyunsaturated fatty acid relative content in cell.This method is compared with conventional fat acid content assay method, extracts, the operation such as transesterification without the need to sample drying, clasmatosis, grease, without the need to an organic solvent, has saved manpower and materials, has substantially increased determination efficiency.
Technical solution of the present invention: a kind of method of content of polyunsaturated fatty acid in quick judgement oil-containing extracellular microbial, comprises the steps.
(1) sample preparation: microbial cell bacterium liquid is carried out centrifugal, by thalline again even suspension in damping fluid, obtains cell suspending liquid; To remove the interference component in nutrient solution outside cell.This step can repeated several times.
(2) magnetic resonance detection: magnetic resonance detection is carried out to cell suspending liquid, obtains nmr spectrum; Described magnetic resonance detection is
1hNMR detect or
13cNMR detects, and described nmr spectrum is
1h spectrum or
13c composes.
(3) result calculates and judges: described in
1in H spectrum, the peak of chemical shift between 1.0-1.3 is peak A, and chemical shift peak between 2.5-2.9 is peak B; Described
13in C spectrum, the peak of chemical shift between 29.0-30.0 is peak A', and the peak of chemical shift between 25.0-26.0 is peak B', and described peak A and peak A' represents saturated fatty acid, and described peak B and peak B' represents polyunsaturated fatty acid; The ratio of described peak B and peak A peak intensity or peak area is R, and the ratio of described peak B' and peak A' peak intensity or peak area is R'; Described R or R' is larger, and the content of polyunsaturated fatty acid is higher.
Preferably, described oil-containing microorganism is store with fat form of dripping the microorganism that grease and fat content are not less than dry cell weight 5%.
Preferably, described oil-containing microorganism comprises fungi, bacterium, micro-algae and yeast.
Preferably, pH and the ionic strength of the damping fluid described in step (1) are close with thalline nutrient culture media.
Beneficial effect of the present invention:
1) the present invention utilizes this feature high of self fat content in cell, direct use cell carries out magnetic resonance detection, sample preparation and testing process are very simple, only need carry out simple substitute to nutrient culture media, without the need to sample drying, grease extracting, the process such as transesterification, without the need to an organic solvent, save manpower and materials.
2) the present invention uses the characteristic peak on nmr spectrum to detect, and uses
1h spectrum or
13c composes.On 600 MHz nuclear magnetic resonance spectrometers of routine,
1h composes and can complete within ten minutes detection time,
13c spectrum can complete within half an hour, and it is convenient rapidly to detect.
3) the present invention is due to rapid and convenient, and the high flux being applicable to a large amount of sample detects, and as microorganism Large-scale Screening, training systern, industrial processes monitoring etc., have important industrialization meaning.
Accompanying drawing explanation
Fig. 1 is schizochytrium cell
1h nmr spectrum.
Fig. 2 is schizochytrium cell
13c nmr spectrum.
Fig. 3 is for using
1the ratio R that H spectrum calculates and use
13c spectrum calculate ratio R ' between relation.
Fig. 4 is that under different condition of culture, schizochytrium uses
1relation between the DHA relative content that H spectrum calculates and the DHA content using GC to measure.
Fig. 5 is that under different condition of culture, schizochytrium uses
13relation between the DHA relative content that C spectrum calculates and the DHA content using GC to measure.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described.
Embodiment 1
The present embodiment is for using more a kind of micro-algae of the inventive method, the content of polyunsaturated fatty acid of schizochytrium under different condition of culture, and concrete steps are as follows:
One, sample preparation: getting each 1 milliliter of 9 schizochytrium cell samples of different condition of culture is centrifugal under the condition of 8000 g at rotating speed, removes supernatant; Bacterial sediment is resuspended in 4 times in the phosphate buffer of thalline volume.Be centrifugal under the condition of 8000 g by this thalline re-suspension liquid at rotating speed, remove supernatant; Bacterial sediment is resuspended in the phosphate buffer equal with thalline volume.The phosphate buffer used in preparation of samples process has the pH identical with nutrient culture media and ionic strength.
Two, magnetic resonance detection: get cell re-suspension liquid 500 microlitre, adds 50 microlitre heavy water, is positioned in the nuclear magnetic resonance sample pipe of 5 mm outer diameter, the nuclear magnetic resonance spectrometer of 600 MHz measures
1h spectrum and
13c composes.
1the pulse protocol of the water peak pressure of H spectrum experiment employing standard,
13c composes use standard
1the pulse protocol of H decoupling.
1it is 3 minutes that H composes experimental period,
13it is 10 minutes that C composes experimental period.Each sample is obtained respectively as Fig. 1
1h spectrum and Fig. 2
13c composes.
Three, result calculates: by calculating the intensity rate R of peak B and peak A or the intensity rate R' of peak B' and peak A', comparing the content of polyunsaturated fatty acid between each sample, the results are shown in Table 1.Known according to table 1, sample 3
1h spectrum R value with
13the R' value of C spectrum is maximum, for containing the highest sample condition of content of polyunsaturated fatty acid.
Four, the checking of this method reliability: method of the present invention is simple, its measuring error is mainly from the impurity that may produce signal in sample at observation spectrum peak position, and the error of nuclear magnetic resonance experiment self.To the sample that we measure
1h spectrum R value with
13c composes R' value and carries out linear regression (see figure 3), its R
2=0.9929, show
1h spectrum and
13c spectrum has close reliability.Grease extraction is carried out to 9 samples measured above, uses GC to measure the content of polyunsaturated fatty acid of each sample.By GC measure result with
1h spectrum and
13c composes the result obtained and carries out linear regression, the results are shown in Figure 4 and Fig. 5, its R
2be 0.8810 and 0.8601, show, between our method and traditional GC method, there is the good goodness of fit.
R and R ' of the sample of table 1. 9 different condition of culture
Claims (4)
1. judge a method for content of polyunsaturated fatty acid in oil-containing microbial cell fast, it is characterized in that: comprise the steps:
Sample preparation: microbial cell bacterium liquid is carried out centrifugal, by thalline again even suspension in damping fluid, obtains cell suspending liquid;
Magnetic resonance detection: magnetic resonance detection is carried out to cell suspending liquid, obtains nmr spectrum; Described magnetic resonance detection is
1hNMR detect or
13cNMR detects, and described nmr spectrum is
1h spectrum or
13c composes;
Result calculates: described in
1in H spectrum, the peak of chemical shift between 1.0-1.3 is peak A, and chemical shift peak between 2.5-2.9 is peak B; Described
13in C spectrum, the peak of chemical shift between 29.0-30.0 is peak A', and the peak of chemical shift between 25.0-26.0 is peak B', and described peak A and peak A' represents saturated fatty acid, and described peak B and peak B' represents polyunsaturated fatty acid; The ratio of described peak B and peak A peak intensity or peak area is R, and the ratio of described peak B' and peak A' peak intensity or peak area is R'; Described R or R' is larger, and the content of polyunsaturated fatty acid is higher.
2. the method for content of polyunsaturated fatty acid in a kind of quick judgement oil-containing microbial cell according to claim 1, is characterized in that: described oil-containing microorganism is store grease with fat form of dripping and fat content is not less than the microorganism of dry cell weight 5%.
3. the method for content of polyunsaturated fatty acid in a kind of quick judgement oil-containing microbial cell according to claim 1, is characterized in that: described oil-containing microorganism comprises fungi, bacterium, micro-algae and yeast.
4., according to the method for content of polyunsaturated fatty acid in a kind of quick judgement oil-containing microbial cell in claim 1-3 described in any one, it is characterized in that: the pH of the damping fluid described in step (1) is consistent with thalline nutrient culture media with ionic strength.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105954308A (en) * | 2016-04-29 | 2016-09-21 | 大连工业大学 | Method for quickly detecting oil content of oil-containing microorganism |
Citations (4)
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|---|---|---|---|---|
| RU2187796C2 (en) * | 2000-08-21 | 2002-08-20 | Витюк Борис Яковлевич | Procedure determining acid number of vegetable oils |
| CN102636510A (en) * | 2011-04-28 | 2012-08-15 | 河南省科学院高新技术研究中心 | Method for detecting quality of edible oil by using hydrogen-nuclear magnetic resonance spectroscopy |
| CN102967616A (en) * | 2012-11-08 | 2013-03-13 | 厦门大学 | Deep sea fish oil identification method |
| US20130071940A1 (en) * | 2011-08-16 | 2013-03-21 | Brown University | Methods of chemoselective derivation of multiple classes of metabolites |
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2014
- 2014-03-27 CN CN201410118153.6A patent/CN104950006B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2187796C2 (en) * | 2000-08-21 | 2002-08-20 | Витюк Борис Яковлевич | Procedure determining acid number of vegetable oils |
| CN102636510A (en) * | 2011-04-28 | 2012-08-15 | 河南省科学院高新技术研究中心 | Method for detecting quality of edible oil by using hydrogen-nuclear magnetic resonance spectroscopy |
| US20130071940A1 (en) * | 2011-08-16 | 2013-03-21 | Brown University | Methods of chemoselective derivation of multiple classes of metabolites |
| CN102967616A (en) * | 2012-11-08 | 2013-03-13 | 厦门大学 | Deep sea fish oil identification method |
Non-Patent Citations (1)
| Title |
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| 陈合等: "《新型食品原料制备技术与应用》", 31 December 2004 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105954308A (en) * | 2016-04-29 | 2016-09-21 | 大连工业大学 | Method for quickly detecting oil content of oil-containing microorganism |
| CN105954308B (en) * | 2016-04-29 | 2018-09-28 | 大连工业大学 | A kind of method of quick detection oil-containing microorganism oil content |
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| CN104950006B (en) | 2017-09-26 |
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