CN106636264A - Method for producing phycoerythrin and polyunsaturated fatty acid by using R. salina - Google Patents
Method for producing phycoerythrin and polyunsaturated fatty acid by using R. salina Download PDFInfo
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Abstract
The invention provides a method for producing phycoerythrin and polyunsaturated fatty acid by using R. salina. The method comprises the steps of culturing the R. salina by using a seed culture medium; inoculating an artificial seawater fermentation medium with the R. salina; culturing the R. salina by using light with different wave lengths and intensities. The method for producing the phycoerythrin and the polyunsaturated fatty acid by using the R. salina increases the substrate utilization rate, and improves the biomass as well as the fermentation level of total protein, the phycoerythrin, lipid and the polyunsaturated fatty acid, thus being very beneficial to promotion of industrial development of producing the phycoerythrin and the polyunsaturated fatty acid by fermenting the R. salina.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to a kind of using the red born of the same parents algae product phycoerythrin of salt life and many
The method of unrighted acid.
Background technology
Parinaric acid (18:4n-3) (SA) be a kind of important ω -3 series polyunsaturated fatty acid (Δ 6,9,12,
15- all-cis formulas-parinaric acid), used as the precursor substance of EPA, SA transformation efficiencies in animal body compare arachidonic acid
It is much higher, the content of internal cholesterol and triglycerides is reduced with auxiliary, promote the effect of internal saturated fat acid metabolic, from
And reduce blood viscosity, promote blood circulation, improve tissue oxygen supply and dispelling fatigue.Fat is prevented in the deposition of vascular wall,
The angiocardiopathies such as formation and development, prevention cerebral thrombus, cerebral hemorrhage, hypertension that prevention of arterial is atherosis.At present SA is main
From vegetable oil (high SA soybean oils) and microalgae.It is low that soybean-source is primarily present SA relative amounts, all by regional limitation etc.
Many problems, so as to cause bad, purifying cost height of smell etc..Conversely, microbial grease (Single Cell Oils, SCO) is no
But large-scale production is easily realized, while also overcome traditional high SA soybean there is a problem of by region and weather conditionality,
Have a extensive future.
Salt is given birth to red born of the same parents algae (Rhodomonas salin or R.salina) and belongs to Pyrenomonadaceae family members, is
Unicellular red algae amphitrichous.Because it is rich in Long carbon chain polyunsaturated fatty acid and natural colouring matter-phycoerythrin, R.salina
The resource algae kind of a great potential it has been considered, at aspects such as food, cosmetics, medicine, scientific research and aquacultures
It is widely used.R.salina synthesis polyunsaturated fatty acid parinaric acid (SA, 18:4n-3) with alpha-linolenic acid (ALA
18:The 20-30.2% and 20.7-29.9% of the total fat of intracellular 3n-3) are accounted for respectively.
The content of the invention
The purpose of this part is to summarize some aspects of embodiments of the invention and briefly introduce some preferable enforcements
Example.May do in this part and the description of the present application summary and denomination of invention a little simplified or omit to avoid making our department
Point, the purpose of specification digest and denomination of invention obscure, and this simplification or omission cannot be used for limiting the scope of the present invention.
In view of above-mentioned and/or existing utilization salt gives birth to red born of the same parents algae (R.salina) production phycoerythrin and polyunsaturated fatty acid
Method exist problem, it is proposed that the present invention.
It is therefore an object of the present invention to provide one kind gives birth to red born of the same parents algae using different wave length and intensity lamp sources autotrophy fermenting salt
(R.salina) method for producing phycoerythrin and polyunsaturated fatty acid, is greatly improved salt and gives birth to red born of the same parents algae (R.salina)
The efficiency of production phycoerythrin and polyunsaturated fatty acid, so as to reduce production cost.
To solve above-mentioned technical problem, the invention provides following technical scheme：One kind is given birth to red born of the same parents algae and produces algae red using salt
The method of albumen and polyunsaturated fatty acid, including using the red born of the same parents algae of seed culture medium culture salt life；Salt is given birth into red born of the same parents algae inoculation
To artificial seawater fermentation medium, using the optical culture salt of different wave length and intensity red born of the same parents algae is given birth to.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The seed culture medium includes glycerine, Na2EDTA、H3BO3、FeCl3、MnSO4、ZnSO4、CoCl2、HCl、
HEPES bufferpH 7.8、Vitamin B12In one or more.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The seed culture medium includes accounting for glycerine, the Na of culture medium quality 10%2EDTA·2H2O 80mM、H3BO3
2mM、FeCl3·6H2O 5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O
0.001mM、HCl 0.1M、HEPES buffer pH 7.8、Vitamin B12 0.3g/L。
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The artificial seawater fermentation medium includes NaCl, MgSO4·7H2O、KCl、NaNO3、CaCl2·2H2O、
KH2PO4、Tricine pH 7.8、NH4Cl、Na2EDTA·2H2O、H3BO3、FeCl3·6H2O、MnSO4·H2O、ZnSO4·
7H2O、CoCl2·6H2O, HCl, HEPES bufferpH 7.8 or Vitamin B12In one or more.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The utilization seed culture medium culture salt gives birth to red born of the same parents algae, and it is that salt is given birth into red born of the same parents algae with 10～50% inoculation
Amount is accessed in seed culture medium, and cultivation temperature is 21～30 DEG C, 40～50h is cultivated under 100～200rpm.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The optical culture of the utilization different wave length and intensity, it is to be in temperature using the light of different wave length and intensity
21～30 DEG C, pH value is 5～9, and Ventilation Rate is 0.01～10m3·min-1, intensity of illumination 50～500 is μm ol m-2s-1's
Under the conditions of, cultivate 5～14 days.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The light of the utilization different wave length and intensity, wherein, the light includes ruddiness, blue light, red blue mixed light, its
It is 15～30cm with the distance of fermentation medium liquid level.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The red blue mixed light, its mixed proportion is blue light：Ruddiness=1:0.1～10.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The red blue mixed light, its mixed proportion is blue light：Ruddiness=1:1～5.
As utilization salt of the present invention give birth to red born of the same parents algae produce phycoerythrin and polyunsaturated fatty acid method it is a kind of excellent
Scheme is selected, wherein：The ruddiness, its wavelength is 590～670nm, and crest is 640nm；The blue light, its wavelength 420～
510nm, crest is 460nm.
The device have the advantages that：
The present invention improves the speed of growth, biomass, the substrate utilization that salt gives birth to thalline when red born of the same parents algae (R.salina) is fermented
Rate, phycoerythrin, total protein, polyunsaturated fatty acid and total lipid content, the speed of growth lifts more than 3 times, and phycoerythrin increases by 8
More than times, total polyunsaturated fatty acid increases by more than 4 times, and biomass increases by more than 7 times, reduces salt and gives birth to red born of the same parents algae
(R.salina) problem for harvesting, reduces the pre-treatment cost that biomass concentration and purpose product are extracted.
Description of the drawings
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below will be to use needed for embodiment description
Accompanying drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for this
For the those of ordinary skill of field, without having to pay creative labor, can be obtaining other according to these accompanying drawings
Accompanying drawing.Wherein：
Fig. 1 is that varying strength white light illumination condition fermenting salt gives birth to red born of the same parents algae (R.salina) growing state signal in the present invention
Figure；
Fig. 2 is that varying strength white light illumination condition fermenting salt gives birth to red born of the same parents algae (R.salina) phycoerythrin synthesis in the present invention
Situation schematic diagram；
Fig. 3 is that varying strength red light irradiation condition fermenting salt gives birth to red born of the same parents algae (R.salina) growing state signal in the present invention
Figure；
Fig. 4 is that varying strength red light irradiation condition fermenting salt gives birth to red born of the same parents algae (R.salina) phycoerythrin synthesis in the present invention
Situation schematic diagram；
Fig. 5 is that varying strength blue light illumination condition fermenting salt gives birth to red born of the same parents algae (R.salina) growing state signal in the present invention
Figure；
Fig. 6 is that varying strength blue light illumination condition fermenting salt gives birth to red born of the same parents algae (R.salina) phycoerythrin synthesis in the present invention
Situation schematic diagram；
Fig. 7 is that the red blue mixing light irradiation condition fermenting salt of varying strength gives birth to red born of the same parents algae (R.salina) growth feelings in the present invention
Condition schematic diagram；
Fig. 8 is that the red blue mixing light irradiation condition fermenting salt of varying strength gives birth to red born of the same parents algae (R.salina) algae red egg in the present invention
White synthesis situation schematic diagram.
Specific embodiment
It is understandable to enable the above objects, features and advantages of the present invention to become apparent from, with reference to specific embodiment pair
The specific embodiment of the present invention is described in detail.
Many details are elaborated in the following description in order to fully understand the present invention, but the present invention can be with
It is different from alternate manner described here to implement using other, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, therefore the present invention is not limited by following public specific embodiment.
Secondly, " one embodiment " or " embodiment " referred to herein is referred to and may be included at least one realization side of the invention
Special characteristic, structure or characteristic in formula." in one embodiment " that in this manual different places occur not refers both to
Same embodiment, nor single or selectively mutually exclusive with other embodiment embodiment.
Embodiment 1：
Salt gives birth to red born of the same parents algae (R.salina) seed culture medium：
Salt gives birth to red born of the same parents algae source seawater, glycerine 10%, Na2EDTA·2H2O 80mM、H3BO3 2mM、FeCl3·6H2O
5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O
50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B12 0.3g/L。
The generation of seed culture two, per generation carry out in liquid amount 50mL/250mL triangular flasks, inoculum concentration 20% (V/V), 23 DEG C,
Shaking table culture 48h under 120rpm.Second generation seed accesses liquid amount 400mL/600mL gas explosion formulas by the inoculum concentration of 20% (V/V)
It is artificial seawater fermentation medium in reactor in reactor, 50～350 μm of ol m of white light intensity-2s-1, fermentation condition is 23
DEG C, Ventilation Rate be 0.1m3Min, ferments 10 days.
Artificial seawater fermentation medium components are, NaCl 310mM, MgSO4·7H2O 10mM、KCl 8mM、
NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·
2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、
CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B12
0.13g/L。
Experimental result is as depicted in figs. 1 and 2：
As a result show：
150 μm of olm of white light intensity-2·s-1Fermenting salt gives birth to red born of the same parents algae (R.salina), reaches maximum biomass 6.71 within 8 days
× 106/mL, phycoerythrin reaches 14.97mg/L, and substrate utilization ratio is 60.5%.Total fat accounts for dry weight 8.56%, and SA accounts for total fat
Fat acid 27.78%.
Wherein, TFA and total fat are determined as follows,
The extraction of TFA：Cell grinding after freeze-drying is uniform, accurately weigh about 1.00g and add 7mL 20%
60min is shaken in HCl, 75 DEG C of water-baths.Then lipid is extracted in three times with 20mL n-hexanes, after rotary evaporation precipitation, blown with nitrogen
Except residual solvent.Weigh and calculate total lipid content.
Total lipid content (%)=m1/m2* 100%
In formula, m1Representative is weighed and obtains TL amount (g), m2Represent lyophilized mycelium quality (g) for lipids extraction.
The measure of aliphatic acid composition：Capillary column (CP Sil-88:0.20 μm of 50.0m × 250 μ m), nitrogen is used as load
Gas, FID is detector.The temperature of injection port is 250 DEG C, and each sampling volume is 1 μ L.Column temperature condition：80 DEG C continue 2min, so
Afterwards 120 DEG C are warmed up to 10 DEG C/min speed, then 180 DEG C are warmed up to 5 DEG C/min, continue 2min, be warmed up to 2 DEG C/min
230 DEG C, last 230 DEG C continue 5min.With the percentage composition that normalization method calculates each aliphatic acid in total fat.
White light intensity is less than 350 μm of ol m-2s-1When, salt give birth to red born of the same parents algae (R.salina) biomass, total lipid content it is high and
Phycoerythrin is directly proportional to intensity of illumination, and white light intensity is higher than 350 μm of ol m-2s-1When salt can be suppressed to give birth to red born of the same parents algae
(R.salina) synthesis of growth, especially phycoerythrin.
Embodiment 2：
Salt gives birth to red born of the same parents algae (R.salina) seed culture medium：
Salt gives birth to red born of the same parents algae source seawater, glycerine 10%, Na2EDTA·2H2O 80mM、H3BO3 2mM、FeCl3·6H2O
5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O
50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B12 0.3g/L。
The generation of seed culture two, per generation carry out in liquid amount 50mL/250mL triangular flasks, inoculum concentration 20% (V/V), 23 DEG C,
Shaking table culture 48h under 120rpm.Second generation seed accesses liquid amount 400mL/600mL gas explosion formulas by the inoculum concentration of 20% (V/V)
It is artificial seawater fermentation medium in reactor in reactor, 50～350 μm of ol m of red light intensity-2s-1, fermentation condition is 23
DEG C, Ventilation Rate be 0.1m3·min-1, ferment 10 days.Wherein, the wavelength of ruddiness is 590～670nm, and crest is 640nm.
Artificial seawater fermentation medium components are, NaCl 310mM, MgSO4·7H2O 10mM、KCl 8mM、
NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·
2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、
CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B12
0.13g/L。
Experimental result is as shown in Figure 3 and Figure 4：
As a result show：
250 μm of ol m of red light intensity-2s-1Fermenting salt gives birth to red born of the same parents algae (R.salina), 10 days up to maximum biomass 1.31 ×
107Individual/mL.150 μm of ol m of red light intensity-2s-1Phycoerythrin reaches when fermenting salt gives birth to red born of the same parents algae (R.salina) 9 days
37.60mg/L, substrate utilization ratio is 75.5%.350 μm of ol m of red light intensity-2s-1Fermenting salt gives birth to red born of the same parents algae (R.salina) 10
It when total fat account for dry weight 11.12%, SA accounts for TFA 30.00% or so.
Wherein, TFA and total fat are determined as follows,
The extraction of TFA：Cell grinding after freeze-drying is uniform, accurately weigh about 1.00g and add 7mL 20%
60min is shaken in HCl, 75 DEG C of water-baths.Then lipid is extracted in three times with 20mL n-hexanes, after rotary evaporation precipitation, blown with nitrogen
Except residual solvent.Weigh and calculate total lipid content.
Total lipid content (%)=m1/m2* 100%
In formula, m1Representative is weighed and obtains TL amount (g), m2Represent lyophilized mycelium quality (g) for lipids extraction.
The measure of aliphatic acid composition：Capillary column (CP Sil-88:0.20 μm of 50.0m × 250 μ m), nitrogen is used as load
Gas, FID is detector.The temperature of injection port is 250 DEG C, and each sampling volume is 1 μ L.Column temperature condition：80 DEG C continue 2min, so
Afterwards 120 DEG C are warmed up to 10 DEG C/min speed, then 180 DEG C are warmed up to 5 DEG C/min, continue 2min, be warmed up to 2 DEG C/min
230 DEG C, last 230 DEG C continue 5min.With the percentage composition that normalization method calculates each aliphatic acid in total fat.
Red light intensity is less than 50 μm of ol m-2s-1When, salt give birth to red born of the same parents algae (R.salina) biomass, total lipid content it is high and
Phycoerythrin synthesis is suppressed.
Embodiment 3：
Salt gives birth to red born of the same parents algae (R.salina) seed culture medium：
Salt gives birth to red born of the same parents algae source seawater, glycerine 10%, Na2EDTA·2H2O 80mM、H3BO3 2mM、FeCl3·6H2O
5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O
50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B12 0.3g/L。
The generation of seed culture two, per generation carry out in liquid amount 50mL/250mL triangular flasks, inoculum concentration 20% (V/V), 23 DEG C,
Shaking table culture 48h under 120rpm.Second generation seed accesses liquid amount 400mL/600mL gas explosion formulas by the inoculum concentration of 20% (V/V)
It is artificial seawater fermentation medium in reactor in reactor, 50～350 μm of ol m of blue light strength-2s-1, fermentation condition is 23
DEG C, Ventilation Rate be 0.1m3·min-1, ferment 10 days.Wherein, the wavelength of blue light is 420～510nm, and crest is 460nm.
Artificial seawater fermentation medium components are, NaCl 310mM, MgSO4·7H2O 10mM、KCl 8mM、
NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·
2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、
CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B12
0.13g/L。
Experimental result is as shown in Figure 5 and Figure 6：
As a result show：
Blue light is more efficient to exciting for the antenna beam in the red born of the same parents algae of salt life, so as to lift the transformation efficiency of chloroplaset,
More energy are provided for cell.250 μm of ol m of blue light strength-2s-1Fermenting salt gives birth to red born of the same parents algae (R.salina), and 10 days up to maximum
Biomass 1.22 × 107Individual/mL, substrate utilization ratio is 75.8%.Phycoerythrin reaches 49.60mg/L, and total fat accounts for dry weight
12.32%, SA account for TFA 31.15%.
Wherein, TFA and total fat are determined as follows,
The extraction of TFA：Cell grinding after freeze-drying is uniform, accurately weigh about 1.00g and add 7mL 20%
60min is shaken in HCl, 75 DEG C of water-baths.Then lipid is extracted in three times with 20mL n-hexanes, after rotary evaporation precipitation, blown with nitrogen
Except residual solvent.Weigh and calculate total lipid content.
Total lipid content (%)=m1/m2* 100%
In formula, m1Representative is weighed and obtains TL amount (g), m2Represent lyophilized mycelium quality (g) for lipids extraction.
The measure of aliphatic acid composition：Capillary column (CP Sil-88:0.20 μm of 50.0m × 250 μ m), nitrogen is used as load
Gas, FID is detector.The temperature of injection port is 250 DEG C, and each sampling volume is 1 μ L.Column temperature condition：80 DEG C continue 2min, so
Afterwards 120 DEG C are warmed up to 10 DEG C/min speed, then 180 DEG C are warmed up to 5 DEG C/min, continue 2min, be warmed up to 2 DEG C/min
230 DEG C, last 230 DEG C continue 5min.With the percentage composition that normalization method calculates each aliphatic acid in total fat.
Blue light strength is less than 50 μm of ol m-2s-1Or higher than 250 μm of ol m-2s-1When, it is biological that salt gives birth to red born of the same parents algae (R.salina)
Amount, total lipid content are high and phycoerythrin synthesis is suppressed.
Embodiment 4：
Salt gives birth to red born of the same parents algae (R.salina) seed culture medium：
Salt gives birth to red born of the same parents algae source seawater, glycerine 10%, Na2EDTA·2H2O 80mM、H3BO3 2mM、FeCl3·6H2O
5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、Na2EDTA·2H2O
50mM、HCl 0.1M、HEPES buffer pH 7.8 10mM、Vitamin B12 0.3g/L。
The generation of seed culture two, per generation carry out in liquid amount 50mL/250mL triangular flasks, inoculum concentration 20% (V/V), 23 DEG C,
Shaking table culture 48h under 120rpm.Second generation seed accesses liquid amount 400mL/600mL gas explosion formulas by the inoculum concentration of 20% (V/V)
It is artificial seawater fermentation medium in reactor in reactor, red blue mixing 50～350 μm of ol m of luminous intensity-2s-1, ferment bar
Part is 23 DEG C, Ventilation Rate is 0.1m3·min-1, ferment 10 days.Wherein, the wavelength of blue light is 420～510nm, and crest is
460nm.Wherein, the wavelength of ruddiness is 590～670nm, and crest is 640nm.In mixed light, the ratio of blue light and ruddiness is 1:3.
Light source is 15cm with the distance of fermentation medium liquid level.
Artificial seawater fermentation medium components are, NaCl 310mM, MgSO4·7H2O 10mM、KCl 8mM、
NaNO312mM、CaCl2·2H2O 2mM、KH2PO40.37mM、Tricine pH 7.8 25mM、NH4Cl0.5mM、Na2EDTA·
2H2O 0.27mM、H3BO31.84mM、FeCl3·6H2O0.018mM、MnSO4·H2O0.097mM、ZnSO4·7H2O0.007mM、
CoCl2·6H2O0.002mM、HCl0.1mM、FeCl3·6H2O3mM、HEPES buffer pH 7.8 10mM、Vitamin B12
0.13g/L。
Experimental result is as shown in Figure 7 and Figure 8：
As a result show：
Red blue mixing 250 μm of ol m of luminous intensity-2s-1Fermenting salt gives birth to red born of the same parents algae (R.salina), reaches maximum biomass within 9 days
1.23×107Individual/mL.Red blue mixing 150 μm of ol m of luminous intensity-2s-1Algae red egg when fermenting salt gives birth to red born of the same parents algae (R.salina) 9 days
53.17mg/L is reached in vain, substrate utilization ratio is 91.8%, total fat accounts for dry weight 14.07%, SA accounts for TFA 26.43%.
Wherein, TFA and total fat are determined as follows,
The extraction of TFA：Cell grinding after freeze-drying is uniform, accurately weigh about 1.00g and add 7mL 20%
60min is shaken in HCl, 75 DEG C of water-baths.Then lipid is extracted in three times with 20mL n-hexanes, after rotary evaporation precipitation, blown with nitrogen
Except residual solvent.Weigh and calculate total lipid content.
Total lipid content (%)=m1/m2* 100%
In formula, m1Representative is weighed and obtains TL amount (g), m2Represent lyophilized mycelium quality (g) for lipids extraction.
The measure of aliphatic acid composition：Capillary column (CP Sil-88:0.20 μm of 50.0m × 250 μ m), nitrogen is used as load
Gas, FID is detector.The temperature of injection port is 250 DEG C, and each sampling volume is 1 μ L.Column temperature condition：80 DEG C continue 2min, so
Afterwards 120 DEG C are warmed up to 10 DEG C/min speed, then 180 DEG C are warmed up to 5 DEG C/min, continue 2min, be warmed up to 2 DEG C/min
230 DEG C, last 230 DEG C continue 5min.With the percentage composition that normalization method calculates each aliphatic acid in total fat.
Red blue mixing luminous intensity is higher than 250 μm of ol m-2s-1When, salt is given birth to red born of the same parents algae (R.salina) phycoerythrin synthesis and is received
To suppression.
Embodiment 5 (comparative example)
Botryococcus braunii is inoculated in the seed culture medium in same embodiment 1～4, cultivated for two generations, per generation is in liquid amount
Carry out in 50mL/250mL triangular flasks, inoculum concentration 20% (V/V), 23 DEG C, shaking table culture 48h under 120rpm.Second generation seed is pressed
The inoculum concentration of 20% (V/V) is accessed in liquid amount 400mL/600mL gas explosion formula reactors, is artificial seawater fermentation training in reactor
Foster base, red blue mixing 50～350 μm of ol m of luminous intensity-2s-1, fermentation condition is 23 DEG C, Ventilation Rate is 0.1m3·min-1, send out
Ferment 10 days.Wherein, the wavelength of blue light is 420～510nm, and crest is 460nm.Wherein, the wavelength of ruddiness be 590～670nm, ripple
Peak is 640nm.In mixed light, the ratio of blue light and ruddiness is 1:3.Light source is 15cm with the distance of fermentation medium liquid level.
Experimental result is, Botryococcus braunii each group poor growth, under red blue mixed light, only in 300～350 μm of ol m- 2s-1There is a small amount of product, indivedual groups hardly grow.
As can be seen here, the present invention improve salt give birth to the speed of growth of thalline when red born of the same parents algae (R.salina) is fermented, biomass,
Phycoerythrin, total protein, polyunsaturated fatty acid and total lipid content, the speed of growth lifts more than 3 times, and phycoerythrin increases by 8 times
More than, total polyunsaturated fatty acid increases by more than 4 times, and biomass increases by more than 7 times, reduces salt and gives birth to red born of the same parents algae (R.salina)
The problem of results, reduces the pre-treatment cost that biomass concentration and purpose product are extracted.
It should be noted that above example is only unrestricted to illustrate technical scheme, although with reference to preferably
Embodiment has been described in detail to the present invention, it will be understood by those within the art that, can be to the technology of the present invention
Scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should cover at this
In the middle of bright right.
Claims (10)
1. a kind of utilization salt gives birth to the method that red born of the same parents algae produces phycoerythrin and polyunsaturated fatty acid, it is characterised in that：Including,
Red born of the same parents algae is given birth to using seed culture medium culture salt；
Salt is given birth into red born of the same parents algae and is inoculated into artificial seawater fermentation medium, using the optical culture salt of different wave length and intensity red born of the same parents are given birth to
Algae.
2. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 1, its feature
It is：The seed culture medium includes glycerine, Na2EDTA、H3BO3、FeCl3、MnSO4、ZnSO4、CoCl2、HCl、HEPES
bufferpH 7.8、Vitamin B12In one or more.
3. utilization salt according to claim 1 or claim 2 gives birth to the method that red born of the same parents algae produces phycoerythrin and polyunsaturated fatty acid, and it is special
Levy and be：The seed culture medium includes accounting for glycerine, the Na of culture medium quality 10%2EDTA·2H2O 80mM、H3BO3 2mM、
FeCl3·6H2O 5.02mM、MnSO4·H2O 0.01mM、ZnSO4·7H2O 0.001mM、CoCl2·6H2O 0.001mM、HCl
0.1M、HEPES buffer pH 7.8、Vitamin B12 0.3g/L。
4. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 1, its feature
It is：The artificial seawater fermentation medium includes NaCl, MgSO4·7H2O、KCl、NaNO3、CaCl2·2H2O、KH2PO4、
Tricine pH 7.8、NH4Cl、Na2EDTA·2H2O、H3BO3、FeCl3·6H2O、MnSO4·H2O、ZnSO4·7H2O、
CoCl2·6H2O, HCl, HEPES bufferpH 7.8 or Vitamin B12In one or more.
5. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 1, its feature
It is：The utilization seed culture medium culture salt gives birth to red born of the same parents algae, and it is that salt is given birth into red born of the same parents algae to access with 10～50% inoculum concentration
In seed culture medium, cultivation temperature is 21～30 DEG C, 40～50h is cultivated under 100～200rpm.
6. give birth to red born of the same parents algae using salt according to any one of claim 1,2,4 or 5 and produce phycoerythrin and polyunsaturated fat
The method of acid, it is characterised in that：The optical culture of the utilization different wave length and intensity, it is the light using different wave length and intensity
It it is 21～30 DEG C in temperature, pH value is 5～9, Ventilation Rate is 0.01～10m3·min-1, intensity of illumination 50～500 is μm ol
m-2s-1Under conditions of, cultivate 5～14 days.
7. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 6, its feature
It is：The light of the utilization different wave length and intensity, wherein, the light includes ruddiness, blue light, red blue mixed light, itself and fermentation
The distance of culture medium liquid level is 15～30cm.
8. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 7, its feature
It is：The red blue mixed light, its mixed proportion is blue light：Ruddiness=1:0.1～10.
9. the method for giving birth to red born of the same parents algae product phycoerythrin and polyunsaturated fatty acid using salt according to claim 8, its feature
It is：The red blue mixed light, its mixed proportion is blue light：Ruddiness=1:1～5.
10. the method that red born of the same parents algae produces phycoerythrin and polyunsaturated fatty acid is given birth to using salt according to claim 7 or 9, its
It is characterised by：The ruddiness, its wavelength is 590～670nm, and crest is 640nm；The blue light, 420～510nm of its wavelength, ripple
Peak is 460nm.
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| CN110923146A (en) * | 2019-10-30 | 2020-03-27 | 海南大学 | Chlorella rich in polyunsaturated fatty acids and essential amino acids and application thereof |
| EP4125420A4 (en) * | 2020-03-31 | 2024-04-17 | V2 Food Pty Ltd | Food colouring agents |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102597255A (en) * | 2009-05-06 | 2012-07-18 | 威克·福雷斯特大学医学院 | Compositions, methods, and kits for polyunsaturated fatty acids from microalgae |
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| CN102597255A (en) * | 2009-05-06 | 2012-07-18 | 威克·福雷斯特大学医学院 | Compositions, methods, and kits for polyunsaturated fatty acids from microalgae |
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| RICARDO M. CHALOUB等: "Combined effects of irradiance，temperature and nitrate concentration on phycoerythrin content in the microalga Rhodomonas sp. (Cryptophyceae)", 《ALGAL RESEARCH》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110923146A (en) * | 2019-10-30 | 2020-03-27 | 海南大学 | Chlorella rich in polyunsaturated fatty acids and essential amino acids and application thereof |
| EP4125420A4 (en) * | 2020-03-31 | 2024-04-17 | V2 Food Pty Ltd | Food colouring agents |
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