CN104950006B - A kind of method of the quick intracellular content of polyunsaturated fatty acid of judgement oil-containing microorganism - Google Patents
A kind of method of the quick intracellular content of polyunsaturated fatty acid of judgement oil-containing microorganism Download PDFInfo
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- CN104950006B CN104950006B CN201410118153.6A CN201410118153A CN104950006B CN 104950006 B CN104950006 B CN 104950006B CN 201410118153 A CN201410118153 A CN 201410118153A CN 104950006 B CN104950006 B CN 104950006B
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Abstract
The invention discloses a kind of method of the intracellular content of polyunsaturated fatty acid of quick detection oil-containing microorganism, the relative amount of intracellular polyunsaturated fatty acid mainly in the microorganism such as detection microalgae, bacterium, fungi, yeast, it is after cell culture fluid is centrifuged, thalline is resuspended in buffer solution, nuclear magnetic resonance is determined1H compose or13C is composed, according to the strength ratio of characteristic peak on nmr spectrum come the content of the polyunsaturated fatty acid of the different samples of comparison.The present invention is compared with conventional fat acid content assay method, without the operation such as sample drying, clasmatosis, grease extraction, transesterification, without using organic solvent, manpower and materials are saved, substantially increase determination efficiency, it is adaptable to the high flux quick detection of a large amount of samples, such as microbe to screen, training systern, commercial process monitoring etc..
Description
Technical field
The invention belongs to detection method technical field, and in particular to a kind of side of intracellular content of polyunsaturated fatty acid
Method, more particularly to it is a kind of suitable for bacterial screening, microculture condition optimizing and the micro- life of industrial production polyunsaturated fatty acid
The method for quickly judging intracellular unsaturated fatty acid content of thing incubation monitoring.
Background technology
Polyunsaturated fatty acid（Mainly include DHA, EPA, ARA etc.）With maintenance Cell Homeostasis and normal growth, prevention
Many important physiological actions such as angiocardiopathy, immunological regulation and anticancer.Polyunsaturated fatty acid is for a long time as baby children
Important additives in youngster's milk powder, health food, medicine are used, and also begin to make in the food such as edible oil, dairy produce in recent years
Its nutritive value is improved for additive.Early stage polyunsaturated fatty acid is mainly derived from deep sea fish oil, dirty by overfishing, environment
The influence of the factors such as dye, seasonal variations is very big.And microbe-derived polyunsaturated fatty acid then have it is with short production cycle, not by
The influence in season and place, not by natural environment pollution effect the features such as, be increasingly becoming substitute deep sea fish oil how unsaturated fat
The main production source of fat acid, and it is widely used in scientific research and industrialized production.Produce micro- life of polyunsaturated fatty acid
Thing includes microalgae, bacterium, fungi, yeast etc., and these microorganisms are generally based on a certain polyunsaturated fatty acid of production；Such as split
Grow kettle algae usual high yield DHA, Shewanella high yield EPA, Mortierella alpina high yield ARA etc..
The microorganism of production polyunsaturated fatty acid generally isolates and purifies from natural environment（Bacterial strain screening）Obtain, and lead to
The methods such as everfermentation engineering carry out medium optimization, training systern and amplification culture etc., eventually for heavy industrialization
Production., it is necessary to polyunsaturated fatty acid in microorganism in bacterial strain screening, training systern, commercial process
Content is measured, to reach the purpose for improving yield or monitor production process.The method for determining aliphatic acid composition at present is usual
Need to extract grease first, then using the methods such as GC, GC-MS, HPLC or NMR determine grease in aliphatic acid composition and contain
Amount.Sample required for these methods usually requires to be prepared by the complicated step such as solvent extraction and derivatization, not only
Time-consuming, and may produce influence to accuracy of detection, causes larger error.Therefore, simple and quick measure is found unsaturated
The method of aliphatic acid, has important for batch samples detection in bacterial strain screening, training systern, commercial process
Meaning.
The content of the invention
It is an object of the invention to provide a kind of method of content of polyunsaturated fatty acid in quick detection microbial cell.
This method high Lipid-producing and can form fat drips this characteristics using microorganism itself, directly using cell progress nuclear magnetic resonance inspection
Survey, by the strength ratio of saturated fatty acid on nmr spectrum and the characteristic peak of unrighted acid, judgement is intracellular more not
Saturated fatty acid relative amount.This method is compared with conventional fat acid content assay method, without sample drying, clasmatosis,
The operation such as grease extraction, transesterification, without using organic solvent, has saved manpower and materials, has substantially increased determination efficiency.
Technical solution of the present invention：A kind of method of quick judgement oil-containing microorganism intracellular content of polyunsaturated fatty acid, bag
Include following steps.
（1）Sample preparation：Microbial cell bacterium solution is centrifuged, by thalline again even suspension in buffer solution, obtained
To cell suspending liquid；To remove the interference component in nutrient solution outside cell.The step may be repeated several times.
（2）Magnetic resonance detection：Magnetic resonance detection is carried out to cell suspending liquid, nmr spectrum is obtained；The core
Magnetic resonance is detected as1HNMR detect or13CNMR detects that the nmr spectrum is1H compose or13C is composed.
（3）As a result calculate and judge：It is described1Peak of the chemical shift between 1.0-1.3 is peak A in H spectrums, and chemical shift exists
2.5-2.9 between peak be peak B；It is described13Peak of the chemical shift between 29.0-30.0 is peak A' in C spectrums, and chemical shift exists
Peak between 25.0-26.0 is peak B', and the peak A and peak A' represent saturated fatty acid, and the peak B and peak B' represent how unsaturated
Aliphatic acid；The ratio of the peak B and peak A peak intensities or peak area is R, the peak B' and peak A' peak intensities or peak area
Ratio is R'；The R or R' are bigger, and the content of polyunsaturated fatty acid is higher.
Preferably, the oil-containing microorganism is with fat drips form storage grease and fat content is not less than dry cell weight
5% microorganism.
Preferably, the oil-containing microorganism includes fungi, bacterium, microalgae and yeast.
Preferably, step（1）The pH and ionic strength of described buffer solution are close with thalline culture medium.
Beneficial effects of the present invention：
1）The present invention directly carries out nuclear magnetic resonance inspection using this high feature of itself intracellular fat content using cell
Survey, sample preparation and detection process are extremely simple, need to only carry out simple substitute to culture medium, extracted without sample drying, grease,
The process such as transesterification, without using organic solvent, saves manpower and materials.
2）The present invention is detected using the characteristic peak on nmr spectrum, is used1H compose or13C is composed.Conventional 600
On MHz nuclear magnetic resonance spectrometers,1H spectrum detection times can be completed within ten minutes,13C spectrums can be completed within half an hour,
Detection is rapid convenient.
3）The present invention is due to rapid and convenient, it is adaptable to the high flux detection of a large amount of samples, such as microorganism Large-scale Screening, training
Condition optimizing, industrial processes monitoring etc. are supported, with important industrialization meaning.
Brief description of the drawings
Fig. 1 is schizochytrium cell1H nmr spectrums.
Fig. 2 is schizochytrium cell13C nmr spectrums.
Fig. 3 is to use1Ratio R and use that H spectrums are calculated13C spectrum calculate ratio R ' between relation.
Fig. 4 uses for schizochytrium under different condition of culture1DHA relative amounts and the DHA using GC measure that H spectrums are calculated
Relation between content.
Fig. 5 uses for schizochytrium under different condition of culture13DHA relative amounts and the DHA using GC measure that C spectrums are calculated
Relation between content.
Embodiment
The invention will be further described below in conjunction with the accompanying drawings.
Embodiment 1
The present embodiment is to use a kind of microalgae of the inventive method, many insatiable hungers of the schizochytrium under different condition of culture
And content of fatty acid, comprise the following steps that：
First, sample preparation：It is 8000 g in rotating speed to take each 1 milliliter of 9 schizochytrium cell samples of different condition of culture
Under conditions of centrifuge, remove supernatant；In the phosphate buffer that bacterial sediment is resuspended in 4 times of thalline volumes.By the thalline
Re-suspension liquid is centrifuged under conditions of rotating speed is 8000 g, removes supernatant；Bacterial sediment is resuspended in equal with thalline volume
In phosphate buffer.The phosphate buffer used during preparation of samples has and culture medium identical pH and ionic strength.
2nd, magnetic resonance detection：500 microlitres of cell re-suspension liquid is taken, 50 microlitres of heavy water is added, is positioned over 5 mm outer diameters
In nuclear magnetic resonance sample pipe, determined on 600 MHz nuclear magnetic resonance chemical analyser1H compose and13C is composed.1H spectrum experiments are using standard
The pulse protocol of water suppression,13C spectrums use standard1The pulse protocol of H decouplings.1H spectrum experimental periods are 3 minutes,13C spectrums are real
The time is tested for 10 minutes.Each sample is respectively obtained such as Fig. 11H is composed with Fig. 2's13C is composed.
3rd, result is calculated：By calculating peak B and peak A intensity rate R or peak B' and peak A' intensity rate R', than
Compared with the content of polyunsaturated fatty acid between each sample, 1 the results are shown in Table.It can be seen from table 1, sample 31H spectrum R values with13C
The R' values of spectrum are maximum, to contain content of polyunsaturated fatty acid highest sample condition.
4th, the checking of this method reliability：The method of the present invention is simple and easy to apply, and its measurement error can in sample
Can observation spectrum peak position produce signal impurity, and nuclear magnetic resonance experiment itself error.The sample that we are determined1H spectrum R values with13C spectrum R' values carry out linear regression（See Fig. 3）, its R2=0.9929, show1H compose and13C spectrums can with close
By property.Grease extraction is carried out to 9 samples above determined, the content of polyunsaturated fatty acid of each sample is determined using GC.
Result that GC is determined with1H compose and13The result that C spectrums are obtained carries out linear regression, as a result sees Fig. 4 and Fig. 5, its R2For 0.8810
With 0.8601, show that there is the preferable goodness of fit between our method and tradition GC methods.
The R and R ' of the sample of the different condition of culture of 1. 9, table
Claims (3)
1. a kind of method of the quick intracellular content of polyunsaturated fatty acid of judgement oil-containing microorganism, it is characterised in that：Including such as
Lower step：
Sample preparation：Microbial cell bacterium solution is centrifuged, by thalline again even suspension in buffer solution, cell is obtained and hangs
Supernatant liquid；The microorganism is the microorganism for being not less than dry cell weight 5% with fat drips form storage grease and fat content；
Magnetic resonance detection：Magnetic resonance detection is carried out to cell suspending liquid, nmr spectrum is obtained；The nuclear magnetic resonance inspection
Survey and be1HNMR detect or13CNMR detects that the nmr spectrum is1H compose or13C is composed；
As a result calculate：It is described1Peak of the chemical shift between 1.0-1.3 is peak A, chemical shift peak between 2.5-2.9 in H spectrums
For peak B；It is described13Peak of the chemical shift between 29.0-30.0 is peak A', peak of the chemical shift between 25.0-26.0 in C spectrums
Saturated fatty acid is represented for peak B', the peak A and peak A', the peak B and peak B' represent polyunsaturated fatty acid；The peak B and
The ratio of peak A peak intensities or peak area is R, and the ratio of the peak B' and peak A' peak intensities or peak area is R'；The R or
R' is bigger, and the content of polyunsaturated fatty acid is higher.
2. a kind of side of quick intracellular content of polyunsaturated fatty acid of judgement oil-containing microorganism according to claim 1
Method, it is characterised in that：The oil-containing microorganism includes fungi, bacterium, microalgae and yeast.
3. the quick judgement intracellular polyunsaturated fat of oil-containing microorganism of one kind according to any one in claim 1-2
The method of acid content, it is characterised in that：The pH and ionic strength of buffer solution described in step (1) are consistent with thalline culture medium.
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