CN109234354A - A kind of screening and coercing cultivation method of high yield PUFAs microalgae - Google Patents
A kind of screening and coercing cultivation method of high yield PUFAs microalgae Download PDFInfo
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Abstract
The present patent application belongs to algae culture technical field, specifically discloses the screening and coercing cultivation method of a kind of high yield PUFAs microalgae, specifically includes following steps, the separation of (1) algae；(2) primary dcreening operation: (3) carry out grease extraction to alternative algae strain and fatty acid component is analyzed, and using the percentage composition of fat content and PUFAs in total fatty acids as standard, filter out the algae strain rich in PUFAs；(4) three nitrogen, phosphorus, iron trophic factors are chosen, coercing cultivation is carried out to the algae strain rich in PUFAs, microalgae fatty acid component is analyzed in the change of measurement frustule growing environment extremely nutritional ingredient；It is separated by grease, measures the variation of different lipid fatty acid components respectively, algae strain change of fat metabolic under the conditions of Nutrient Stress is analyzed.This method can filter out the microalgae with high PUFAs yield, and by coercing micro algae growth, so that microalgae cell is resisted stress and is started self-defence system, to generate higher PUFAs accumulation.
Description
Technical field
The invention belongs to algae culture technical fields, specifically disclose the screening and stress training of a kind of high yield PUFAs microalgae
The method of supporting.
Background technique
Fatty acid is long aliphatic hydrocarbon chain, different types of fatty acid length, saturation degree and in terms of
All difference.Compared to saturated fatty acid and monounsaturated fatty acids, containing there are two and more than two unsaturated bonds more insatiable hungers
There is even more important physiological action to body with fatty acid (PUFAs).
PUFAs is broadly divided into ω -3 series fatty acid and ω -6 series fatty acid, wherein apart from hydrogen-oxygen chain carboxyl distalmost end
Double bond be ω -3 series fatty acid between 3rd, 4 carbon atom reciprocal, the double bond apart from hydrogen-oxygen chain carboxyl distalmost end exists
It is ω -6 series fatty acid between 6th, 7 carbon atom reciprocal.
Microalgae is the high-quality source of PUFAs, a variety of essential amino acids rich in needed by human body.In fact, people open already
Begin using PUFAs abundant in microalgae, such as in aquaculture, fish, shellfish and software are improved by manually adding microalgae
PUFAs (the especially content of ω -3 Series P UFAs) in animal.Although the existing commercialized product of microalgae PUFAs at present,
It is to only have several seeds algaes to be used to produce at present, and only a small number of enterprises have successfully knowhow and key technology.
Microalgae biomass accumulation is lower, algae PUFAs low yield and processing technology it is immature be the key that current to need to break through to ask
Topic.
Really realize that the microalgae of large-scale commercial culture only has a few at present, predominantly spirulina, chlorella, Du
Family name's salt algae, nostoc and synnema algae, stringent safety evaluation limit the advantage of microalgae bio-diversity.In addition, current
Under microdisk electrode technical conditions, some valuable algaes are also difficult to realize high yield.
Summary of the invention
The purpose of the present invention is to provide one kind can generate higher PUFAs accumulation high yield PUFAs microalgae screening and
Coercing cultivation method.
In order to achieve the above object, base case of the invention are as follows:
A kind of screening and coercing cultivation method of high yield PUFAs microalgae, specifically include following steps,
(1) algae separates: acquisition soil sample and water sample carry out algae separation with BG-11 culture medium and f/2 culture medium respectively；
(2) primary dcreening operation: Nile red decoration method carries out primary dcreening operation to isolated algae strain, filters out the high algae strain of grease relative amount；
(3) grease extraction is carried out to alternative algae strain and fatty acid component is analyzed, with fat content and PUFAs in total fatty acids
In percentage composition be standard, filter out rich in PUFAs algae strain；
(4) three nitrogen, phosphorus, iron trophic factors are chosen, coercing cultivation is carried out to the algae strain rich in PUFAs, measurement frustule is raw
The change of long environment extremely nutritional ingredient, analyzes microalgae fatty acid component；It is separated by grease, measures different rouge respectively
Algae strain change of fat metabolic under the conditions of Nutrient Stress is analyzed in the variation of class fatty acid component.
The working principle and beneficial effect of this base case are:
BG-11 culture medium is widely used in the culture of freshwater microalgae, and f/2 culture medium is widely used in the training of marine algae
It supports；Nile red dyeing is a kind of widely used lipophilic oxazines class fluorescent dye, it can be with intracellular various ingredients knot
It closes；This method can filter out the microalgae with high PUFAs yield, and by coercing micro algae growth process, make micro-
Frustule is to resist stress and start self-defence system, to make the entire metabolism network reprogramming of microalgae cell, is generated higher
PUFAs accumulation.
Further, the soil sample of acquisition and water sample need to carry out removal of impurities processing, and soil sample need to remove macroscopic stone and plant
Object root, stem and leaf, water sample need to remove macroplankton and suspended particulate, can guarantee experimental result through 250 mesh silk cover filterings
Accuracy.
Further, the grease extraction are as follows: weigh algae powder in centrifuge tube, in chloroform, methanol and aqueous systems into
Row frustule is crushed and vortex oscillation, is divided into system two layers, and lower layer's oxygen is collected after centrifugation and imitates phase.It is added in the system of upper layer
1ml chloroform repeats vortex oscillation and centrifugally operated, extracting solution is merged, is dried with nitrogen, the component remained in centrifuge tube is
Grease.Relatively simple quickly the grease in algae powder can be extracted.
Further, coercing cultivation is carried out using the method for subsection filter, O-7 days are normal growth stage, and 7-14 days are the side of body
Compel cultivation stage, the content improvement effect of the microalgae after coercing using this method, PUFAs is preferable.
Further, fatty acid component measurement includes being measured to total rouge, neutral fats, glycolipid, phosphatide, can more comprehensively
Various fatty acid components are measured, ensure that the accuracy of data result.
Specific embodiment
Below by specific embodiment, the present invention is described in further detail:
Embodiment
A kind of screening and coercing cultivation method of high yield PUFAs microalgae, specifically include following steps,
(1) soil sample 2g is acquired, (soil sample and water sample of acquisition need to carry out removal of impurities processing to water sample 5ml, and soil sample need to remove naked eyes
Visible stone and plant roots cauline leaf, water sample need to remove macroplankton and suspended particulate through 250 mesh silk cover filterings).
Every kind of sample is cultivated with two kinds of culture mediums of BG-11 and f/2 culture medium respectively, and culture medium additive amount is 20ml,
It is inoculated in 50ml conical flask, is placed in illumination cultivation and cultivates, condition of culture is 28 DEG C, periodicity of illumination 24:0, light intensity 2000-
3000lux is shaken every day conical flask twice, carries out microscopy observation to sample culturing liquid within each two days.
1ml culture solution is taken, first with 500 mesh silk cover filterings, takes a drop Clarified Culture Fluid in the glass that on glass slide, closes the lid later
Piece is placed in the type and quantity of object microscopic observation frustule, and when frustule quantity is more in the visual field, enrichment terminates, sample
Culture solution starts to carry out the separation and screening of microalgae.
(2) primary dcreening operation: Nile red decoration method carries out quick primary dcreening operation to isolated algae strain, filters out the high algae of grease relative amount
Strain.The specific method is as follows: 1. from being selected on solid plate after single algae is inoculated in and cultivates one week or so in BG-11 culture medium, Shi Zao
Liquid OD680Between 0.1-0.4；2. adding 200ul algae solution into transparent 96 orifice plate, it is put into fluorescence microplate reader and detects OD680；③
Into 96 orifice plate of black non transparent be added 198ul algae solution, using multi-function microplate reader carry out fluoremetry, excitation wavelength 488nm,
Launch wavelength 590nm, measures fluorescent value.4. the 0.1mgml of 2ul is added in 96 orifice plates in 3.-1Nile red dye liquor mixes
It is even, it is mixed in 37 DEG C, Incubation in dark 10min, revolving speed 120rmp in 37 DEG C of shaking tables.In multi-function microplate reader again later
It is measured, obtains fluorescent value.Nile red dye acetone solution is divided in 1.5ml centrifuge tube, is saved in 20 DEG C；⑤
The opposite highest 10 plants of algaes of fat content are finally selected according to measurement result carries out programmed screening.6. alternative algae strain expands training
It supports: 10 plants of algaes that Nile red decoration method filters out is carried out with the expansion culture of 1L system, logarithmic phase is inoculated with, inoculative proportion 1:
50, cultivation temperature is 28 DEG C, intensity of illumination 2000-3000lux, light dark period 24:0, is passed through the filtrated air of 2%C02, is trained
After supporting 7 days, by being collected by centrifugation frustule, after -80 DEG C of pre-freezes, 48h is handled in freeze drier, dry algae powder is made.
It is as follows that grease extracts primary operational: weighing 0.59 algae powder in 50mL centrifuge tube, 9ml chloroform/methanol/water body is added
It is (VChloroform: VMethanol: VWater=1:2:0.8), frustule is carried out using ultrasonic cell disintegration instrument and is crushed, and 5ml is added in system later
Chloroform and 5ml water, make V in systemChloroform: VMethanol: VWaterSystem can be observed at this time and be obviously divided into by=1:1:0.9, vortex oscillation 1min
Two layers.It is centrifuged in centrifuge and collects lower layer's oxygen and imitate phase.In the system of upper layer be added 1ml chloroform, repeat vortex oscillation and from
Heart operation, extracting solution is merged, is dried with nitrogen, the component remained in centrifuge tube is grease.
(3) fatty acid component measures 1. methyl esterification of fatty acid: carrying out esterification processing to fatty acid using boron trifluoride method.
2. gas chromatography mass spectrometry (GC-MS) detects fatty acid component.
(4) three nitrogen, phosphorus, iron trophic factors are chosen, coercing cultivation is carried out to algae strain, separated, measured respectively not by grease
With the variation of lipid fatty acid component, algae strain change of fat metabolic under the conditions of Nutrient Stress is analyzed.
This experiment carries out coercing cultivation using the method for subsection filter.O-7 days are normal growth stage, use standard f/
2 culture mediums cultivate algae strain, to expand algae cell density.Cultivating system is IL, intensity of illumination 2000-3000lux, temperature
28 DEG C, it is passed through 2%CO2Filtrated air, every group three groups of setting are parallel.7-14 days are the coercing cultivation stage, according to the battalion of selection
It supports the different of stress factors and changes medium component.This patent chooses N, P and Fe as the Nutrient Stress factor, concrete scheme such as table
1。
Table 1
It is measured during coercing cultivation using following growing state of the cell counting to algae strain S7-M11.
1. the measurement of chlorophyll fluorescence parameters Fv/Fm, reflection frustule physiological status is common when chlorophyll fluorescence parameters
Characteristic value, this tests the measurement that the index is carried out using photosynthesis physiological target FMT150.The green rope luminoscope of leaf built in it
It is able to carry out the inspection side of the green rope fluorescence of micro- bath leaf.Concrete operations are as follows: shaking up algae solution before measurement every time, sample under aseptic condition
Algae solution is carried out dark adaptation 15min by 20ml.Algae solution is transferred in the culture pond of bioreactor later, did not had algae solution internal
The light hole of chlorophyll fluorescence instrument, barn door is put well.The value of Fv/Fm can be directly read by instrumentation later.
2. total sugar content measurement-Phenol sulfuric acid procedure is carried out using sugared content of traditional sugared content measuring method to microalgae
Detection, the method is as follows: 20mg dry algae powder is weighed in 50mL centrifuge tube, addition 20ml water, 5 minutes ultrasonic (output power 30%,
Work 1s, interval 3s), keep algae powder evenly dispersed in water, system is diluted four times.System is added to 10ml after taking 2mL to dilute
In colorimetric cylinder, 6% phenol solution 1.0ml and concentrated sulfuric acid 5.0mL is added, shakes up cooling, 30 DEG C of water-bath 30min of constant temperature, measurement
49Onm absorbance presses same color operation as blank using 2.0 water.
3. total protein content measures, steps are as follows for specific experiment, weighs 20mg algae powder first and is centrifuged in 50ml；It is added in pipe
In the NaOH solution of 10ml0.5mol/L, 10min is extracted in 80 DEG C of water-baths, is stirred.It is quickly cooled to room temperature, is centrifuged
Supernatant is transferred in 25ml volumetric flask by 5000rpm, 5min.It is repeated once extraction.Collect supernatant, constant volume.Then BCA is used
The measurement of kit progress protein concentration.SolutionA is rocked into mixing when concrete operations use as follows, is measured as needed
Sample size, add 1 volume solutionB (50:1) to prepare a certain amount of BCA working solution, mixing by 50 volume solutionA
Uniformly.The BCA working solution prepared can be reserved for for 24 hours at room temperature, is completely dissolved protein standard substance (5mg/ml, BSA), takes
10ul is diluted to 100ul, concentration 0.5mol/L with the Na0H solution of 0.5mol/L.Standard items 0.5mg/ml presses 0 after diluting,
1,2,4,8,12,16,20ul is each sequentially added in 96 orifice plates, and is supplemented to 20ul with standard dilutions, while distinguishing again
The sample to be tested of 20ul is added into corresponding sample well.200ulBCA working solution is added in each hole, is gently blown and beaten with sample loading gun mixed
It is even, after 60 DEG C of placement 30min are cooled to room temperature, the absorbance under 562nm is measured with microplate reader.With the standard concentration read
Value draws standard curve, and obtains the protein concentration of sample to be tested by standard curve with light absorption value.
4. fat content measures, specific method fat content measuring method as used above.
5. 3ml chloroform is added in the algae oil extracted for grease separation method, draws 2ml and carry out grease separation, use solid phase
Extraction pillar separates grease, and sample is added in solid phase extraction column, first elutes neutral fats with 1ml chloroform, then use
10ml acetone elutes glycolipid, finally elutes phosphatide with 10ml methanol, after nitrogen is blown, measures containing for three kinds of lipids by weighing
Amount.
6. fatty acid component measures, by test method as above to total rouge, neutral fats, glycolipid, phosphatide progress fatty acid
Compound mensuration.
Conclusion
(1) influence of the N stress to algae strain S7-M11 lipid metaboli
Under normal culture in O-7 days, algae cell density has reached 2.62 × 106A mL-1.Start to coerce within 7th day, Stress treatment
Front and back is measured cell density, three groups show Stress treatment after algae cell density have a degree of decline, this
It is due to being centrifuged algae solution, the process for cleaning algal gel results in the loss of a small amount of frustule.During coercing cultivation, with control group phase
It is more more vigorous than the growth of, N+ group, final cell density improve 7.21%, N+ group Fv/Fm be stably held in 0.6-0.7 it
Between, a little higher than control group, this illustrates that frustule physiological status keeps good.N- group cell density is presented after of short duration raising and is held
Continuous downward trend, eventually reduces 69.33%, Fv/Fm value compared to the control group and occurs apparent decline compared to the control group
Trend, this shows that frustule photosynthetic efficiency persistently reduces, and growth receives stress from outside, it is difficult to maintain normal physiology shape
State.
Influence of the N stress to algae strain S7-M11 biochemical component intracellular, under coercing cultivation, algae three big energetic supersession objects of strain contain
Significant change has occurred in amount, compared with the control group, N- group carbohydrate content and total lipid content be respectively increased to 10.34% and
31.62%, total protein content drops to 18.00%；On the contrary, N+ group carbohydrate declines with total lipid content, and total protein
Content rises to 34.37%.The variation tendency of three big energy matters is consistent with cell physiological state.Microalgae is in reply nitrogen stress stress
When, some tend to accumulate carbohydrate, and some can more be accumulated, and for algae strain S7-M11, under the conditions of nitrogen stress,
17.22% and 21.03% has been respectively increased in carbohydrate content and fat content, this illustrates that S7-M11 own metabolism is responding
When without N stress, carbon metabolism flow can more flow to oil and fat accumulation.
Influence of the N stress to algae strain S7-M11 total rouge fatty acid composition, under N stress condition of culture, total rouge of S7-M11
Fatty acid component is substantially change.In N+ group, with C14:0 and C16:0, the SFA based on C18:0 accounts for total fatty acids
40.13% reduces by 6.80% compared with the control group, wherein C16:O reduce the content of SFA in 25.82% 1 group then it is significantly raised extremely
61.11% (control group 42.8%) rises 42.83%, and C14:0, C16:0 and C18:0 content significantly increase.N+ group with
Monounsaturated fatty acids total amount variation based on C16:1 and C18:1 is little, and apparent reduce is presented in N- group.PUFAs and C18:
Based on the long-chains PUFAs such as the short chain PUFAs such as 2 and C18:3 and C20:3, C20:4, C20:5 and C20:6.N+ group PUFAs total hundred
Divide and increase 12.17% than content, is mainly shown as raising and C18:3, C20 of the W-3 Series P such as C18:2, C20:4 UFAs:
3, the raising of the W-3 Series P such as C20:5 and C22:6 UFAs.N- group PUFAs percentage composition declines 27.88%, wherein C18:
3, C20:3 and C20:4 is by being substantially reduced.
This time C18:3 is valence linolenic acid in measurement, and C20:4 is arachidonic acid, C20:5 EPA, C22:6 DHA, this
A little PUFAS, which are retransmitted in organism, waves important function, belongs to the nutriment with high added value.According to above data point
Analysis reduces it is found that having under nitrogen sufficiency and synthesizing these functionality PUFAs degrees slightly using S7-M11, makes its application
Value Area is in the development and utilization of nutriment.And nitrogen stress handles the saturated fatty acid that S7-M11 can be promoted to synthesize short chain, these
Fatty acid is easily used to production biodiesel since its saturation degree height, property are stablized, and therefore, it is raw that nitrogen stress condition is conducive to algae strain
The development and utilization of object energy field.
(4) variation of all kinds of lipid fatty acid compositions of algae strain S7-M11: this experiment passes through grease Separation Research grease
Variation of the ingredient in stress procedure.The experimental results showed that N+ group content of phospholipid increases 36.10%, when this shows nitrogen abundance,
Cell division is vigorous, needs to synthesize more phosphatide for vital movement.N- group neutral fats has apparent increase, compared to control
21.13%, the N- group neutral fats content of group neutral fats content is up to 31.45% and improves 48.82%, neutral fats SFA content compared with
More, this result can also explain the phenomenon that N- group SFA significantly increases in experimental result.Meanwhile N- group glycolipid content also has obviously
Increase, thus it is speculated that the reason of this phenomenon occurs may be cell in nitrogen-free pressure starting defense mechanism, this process needs more polysaccharide
Rouge plays a role.
After rouge separation, the fatty acid component of NL, GL and PL are measured respectively, further inquired into different under stress conditions
The Variation Features of lipid fatty acid composition.It is learnt from the analysis of total rouge fatty acid component, N+ condition can promote frustule to accumulate
PUFAs, especially tri- kinds of important PUFAs of C18:3, C20:4, C22:6 are significantly increased, and come from all kinds of lipid fatty acids composition
The C18:3 for seeing that the raising of C18:3 percentage composition is mainly reflected in GL increases, and C20:4 percentage composition in three kinds of lipids has
Increase, C22:6 is mainly reflected in percentage composition in PL and increased significantly.Although the composition ratio of C20:5 omits in total rouge under the conditions of N+
Lower than control group, but significantly improved in GL.In addition, for distribution of certain fatty acid component in three lipoids into
Row analysis, it can be deduced that, the SFA and MUFA of rich content are primarily present in NL in the frustules such as C16:O, C16:O, and C18:
3, C20:4, C22:5 are that the PUFAs of representative is present in GL and PL isopolarity rouge more.This result further confirmed NL, GL,
Based on energy storage, GL and PL then belong to functional lipid by PL and in vivo respective one NL of physiological function, participate in more
Kind vital movement.
(2) influence of the P deficiency to algae strain S7-M11 lipid metaboli
1. influence of the P deficiency to algae strain S7-M11 growth, experimental result are shown.Cell density is consistently higher than under the conditions of P+
Control group, and it is convergent in stress latter stage and control group, and this result illustrates the somatotrophic work of P+ conditions on cell compared with N+ group
With being weaker than N+ condition.Although and P- group cell density always below control group, its decline degree (17.42%) and N- group
(69.31%) it compares, it is even more serious to the growth effect of frustule than phosphorus shortage also to illustrate that nitrogen lacks, as a result also confirms simultaneously
, obviously because of nutritional deficiency, its physiological status changes the variation P- group cell of Fv/Fm, and value is reduced to 0.49, is higher than N-
Group (0.37).P+ group Fv/Fm parameter keeps stablizing, and illustrates that frustule physiological status is good.
2. influence of the P deficiency to algae strain S7-M11 biochemical component intracellular
Under the conditions of P+, fat content has dropped 20.82%, and carbohydrate content is held essentially constant, total protein content
Increasing 14.7% combines growth curve to analyze, and shows that sufficient phosphorus promotes the growth division of cell to a certain extent.P- condition
Under, total protein content decline 50.01%, total lipid content increases 9.44%.Similar with N stress, it is thin that phosphorus shortage equally promotes algae
Born of the same parents accumulate grease, and lacking for nutrient causes cell growth to be suppressed, and protein synthesis is reduced.
3. influence of the P deficiency to the total rouge fatty acid composition of algae strain S7-M11
Under P deficiency condition of culture, total rouge fatty acid component of S7-M11 is substantially change, and phosphorus sufficiency is main
The accumulation of MUFA is promoted, MUFA percent of total improves 16.34%.It is embodied in C16:1 percentage and improves 5.61%,
C18:1 percentage improves 54.90%, and short chain PUFAs also has to be increased to a certain degree, and C18:2 percentage improves 24.53%, C18:3
Percentage improves 8.55%.Under the conditions of P-, SFA percentage composition increases 25.51%, C14:0, the percentage composition of C16:0, C18:0
60.27% is promoted respectively.16.22%, 118.07%, on the whole, P- condition effectively promotes S7-Mll frustule saturated fat
The accumulation of fat acid, while the percentage composition of C20:5 and C22:5 are improved, and P+ condition has been chiefly to facilitate the accumulation of MUFA.
4. the variation of all kinds of lipid fatty acid compositions of algae strain S7-M11
The result shows that frustule GL content increases 12.90%, and PL content reduces compared to the control group under the conditions of P+
112.47%.NL content reduces by 2.42%.Under the conditions of P-, NL increases 17.74% compared to the control group, has dropped 38,91%.PL
Increase 27.35%.
After rouge separation, the fatty acid component of NL, GL and PL are measured respectively, further under the conditions of discussion P deficiency not
With the Variation Features of lipid fatty acid composition.It is in particular in three kinds of lipids, C16:0 and C18:0 have different degrees of liter
It is high.No matter PUFAs does not show advantage in GL, and the content of C22:5 and C22:6 has from GL under the conditions of P+ or P-
To the phenomenon that PL transfer.
(3) influence of the excessive Fe2+ to algae strain S7-M11 lipid metaboli
1. excessive Fe2+ shows that Fe+ group cell density is higher than control group to the influence experimental result of algae strain S7-M11 growth.And
Fe- group cell density is lower than control group, and the shortage of ferro element causes inhibition to cell growth.Ferro element participates in frustule
The formation of Photosystem I and Photosystem I I plays an important role in photosynthesis, respiration and DNA synthesis process, lacks
Under the conditions of iron, frustule is difficult to maintain normal physiological activity.Fe- class value Fv/Fm value is dropped from 0.61 when starting stress is lasting
As low as 0.47, also illustrate that iron deficiency keeps frustule physiological status continuous worsening.For Fe+ group, Fv/Fm value wave between 0.6-0.7
It is dynamic, it is consistent with control group, illustrates that this group of frustule physiological status keeps good.
2. influence of the excessive Fe2+ to algae strain S7-M11 biochemical component intracellular: compared with the control group, under iron sufficiency, algae is thin
The total protein content and total lipid content of born of the same parents is improved, and 22.13% and 10.05% has been respectively increased, and carbohydrate accounts for dry weight
Percentage reduces 31.72%, and under the conditions of nitrogen stress, there is the content of carbohydrate in frustule is dramatically increased, and accounts for dry weight
Percentage is 2.15 times of control group, and the percentage that albumen and total rouge account for dry weight reduces 26.73% and 8.28% respectively.This
The result shows that high excessive Fe2+ can promote algae strain S7-M11 accumulation grease, and under the conditions of Fe Deficiency, the defence machine of frustule
System preferentially selects synthetic carbohydrate for energy reserve, and carbon metabolism flow more flows to the accumulation of carbohydrate.
3. influence of the excessive Fe2+ to the total rouge fatty acid composition of algae strain S7-M11: the algae strain total rouge rouge of S7-M11 under the conditions of N stress
The variation of fat acid constituents is as follows, and compared with the control group, Fe+ group SFA percentage composition increases 12.43%, is embodied in C16:0
With the rising of two kinds of SFA percentage compositions of C18:0.Its SFA percentage composition of Fe- reduces by 23.36%, MUFA and is basically unchanged.Fe- group
SFA percentage composition, which reduces by 17.75%, MUFA percentage composition, reduces by 5.91%, PUFA percentage composition raising 26.84%, main body
The raising of present tri- kinds of fatty acid percentage compositions of C18:3, C20:4 and C20:5, respectively the 1.99 of control group times, 1.11 times and
1.37 again.From the point of view of the variation of the percentage composition of C22:6 and its precursor substance C22:5, two kinds of conditions of high-speed rail and iron-free are not suitable for
Promote frustule accumulation DHA high-speed rail condition that can promote frustule growth and its accumulation of grease, comes from fatty acid component variation
It sees, high-speed rail condition suitably facilitates the synthesis of saturated fatty acid, therefore for algae strain S7-M11, high-speed rail stress conditions are more conducive to
It is developed in the application of field of biological energy source.Iron deficiency condition can promote linolenic acid in algae strain S7-M11, arachidonic acid and EPA's
Synthesis is conducive to develop its nutritive value, but iron deficiency condition will lead to cell that growth is suppressed, and reduces yield of biomass.
4. the variation of all kinds of lipid fatty acid compositions of algae strain 57-: under excessive Fe2+ training method, control group, high-speed rail group, iron deficiency
Group rouge separating resulting is as follows, and NL, GL and PL show more regular changes of contents with the difference of condition of culture.With control group phase
Compare, Fe+ group NL dramatically increases 30.34%, and GL and PL isopolarity rouge reduces (reduces 41.92% He respectively
47.66%).This shows that high excessive Fe2+ can be conducive to S7-M11 and promote synthesis neutral fats, and SFA and MUFA content is rich in neutral fats
It is rich.The changes of contents and Fe- group of three kinds of lipids of Fe- group increase 21.36%, PL on the contrary, its NL content reduces by 9.31%, GL content
Content is held essentially constant.Iron deficiency processing increases the polar lipid content in algae strain S7-M11 cell based on GL.
In conjunction with the further algae strain S7-M11 lipid metaboli under the conditions of research excessive Fe2+ of variation of NL, GL and PL fatty acid component
Variation.Learn that Fe+ condition can promote S7-M11 frustule to accumulate SFA, and wherein C16:0 and C18:O has aobvious from the above analysis
It writes and improves.Meanwhile under the conditions of Fe+, C18:3 and C20:5 show the trend concentrated from NL, PL to GL.Under the conditions of Fe-,
C18:3 percentage composition in neutral fats is held essentially constant, but proportion is significantly increased in GL and PL.C20:5's
Variation is similar under the conditions of Fe+, significantly improves also in the content in GL.And for Fe+ and Fe- condition, C22:5 and C22:
The phenomenon that 6 presentation is shifted from GL into PL.
What has been described above is only an embodiment of the present invention, and the common sense such as well known specific structure and characteristic are not made herein in scheme
Excessive description.It, without departing from the structure of the invention, can be with it should be pointed out that for those skilled in the art
Several modifications and improvements are made, these also should be considered as protection scope of the present invention, these all will not influence what the present invention was implemented
Effect and patent practicability.
Claims (5)
1. the screening and coercing cultivation method of a kind of high yield PUFAs microalgae, which is characterized in that following steps are specifically included,
(1) algae separates: acquisition soil sample and water sample carry out algae separation with BG-11 culture medium and f/2 culture medium respectively；
(2) primary dcreening operation: Nile red decoration method carries out primary dcreening operation to isolated algae strain, filters out the high algae strain of grease relative amount；
(3) grease extraction is carried out to alternative algae strain and fatty acid component is analyzed, with fat content and PUFAs in total fatty acids
Percentage composition is standard, filters out the algae strain rich in PUFAs；
(4) three nitrogen, phosphorus, iron trophic factors are chosen, coercing cultivation is carried out to the algae strain rich in PUFAs, measure frustule growth ring
The change of border extremely nutritional ingredient, analyzes microalgae fatty acid component；It is separated by grease, measures different lipid rouge respectively
Algae strain change of fat metabolic under the conditions of Nutrient Stress is analyzed in the variation of fat acid constituents.
2. a kind of screening and coercing cultivation method of high yield PUFAs microalgae as described in claim 1, which is characterized in that acquisition
Soil sample and water sample need to carry out removal of impurities processing, soil sample need to remove macroscopic stone and plant roots cauline leaf, and water sample need to be through 250
Mesh silk cover filtering removes macroplankton and suspended particulate.
3. a kind of screening and coercing cultivation method of high yield PUFAs microalgae as described in claim 1, which is characterized in that described
Grease extraction are as follows: weigh algae powder in centrifuge tube, frustule is carried out in chloroform, methanol and aqueous systems and is crushed and the vibration that is vortexed
It swings, is divided into system two layers, lower layer's oxygen is collected after centrifugation and imitates phase.In the system of upper layer be added 1ml chloroform, repeat vortex oscillation and
Centrifugally operated merges extracting solution, is dried with nitrogen, and the component remained in centrifuge tube is grease.
4. a kind of screening and coercing cultivation method of high yield PUFAs microalgae as described in claim 1, which is characterized in that use
The method of subsection filter carries out coercing cultivation, and O-7 days are normal growth stage, and 7-14 days are the coercing cultivation stage.
5. a kind of screening and coercing cultivation method of high yield PUFAs microalgae as described in claim 1, which is characterized in that fat
Acid constituents measurement includes being measured to total rouge, neutral fats, glycolipid, phosphatide.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484531A (en) * | 2019-09-02 | 2019-11-22 | 浙江海洋大学 | A kind of screening technique of oil-rich microalgae |
| CN111172039A (en) * | 2020-03-10 | 2020-05-19 | 宁波大学 | Two-stage low-nitrogen low-phosphorus stress microalgae culture method |
-
2018
- 2018-09-10 CN CN201811049582.7A patent/CN109234354A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| BAJWA ET AL.: "Evaluation of Nutrient Stress (Nitrogen,Phosphorus Regimes) on Physio-Biochemical Parameters of Oleaginous Micro algal Strains and SEM Study under Nutrient Stress.", 《INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES & NATURAL RESOURCES》 * |
| YU ET AL.: "Chemicals to enhance microalgal growth and accumulation of high-value bioproducts.", 《FRONTIERS IN MICROBIOLOGY》 * |
| 李雨晨: "高产PUFAs微藻的筛选、营养胁迫及其在鸡饲料中应用的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484531A (en) * | 2019-09-02 | 2019-11-22 | 浙江海洋大学 | A kind of screening technique of oil-rich microalgae |
| CN111172039A (en) * | 2020-03-10 | 2020-05-19 | 宁波大学 | Two-stage low-nitrogen low-phosphorus stress microalgae culture method |
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