CN105331571B - A kind of selection and a kind of schizochytrium limacinum of schizochytrium limacinum - Google Patents
A kind of selection and a kind of schizochytrium limacinum of schizochytrium limacinum Download PDFInfo
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Abstract
The present invention provides a kind of selections of schizochytrium limacinum, and the bacteria suspension of the initial thallus of schizochytrium limacinum is prepared including (1)；(2) bacteria suspension is coated on bagasse hydrolyzate solid medium and is cultivated；(3) single colonie survived on the bagasse hydrolyzate solid medium is selected, respectively secondary culture；(4) selection can stablize passage and the high bacterial strain of thallus DHA yield.Selection of the invention is simple and fast, low in cost, and the bacterial strain of breeding can be grown in non-detoxification bagasse hydrolyzate, and DHA yield is high.
Description
Technical field
The present invention relates to a kind of selection of schizochytrium limacinum and a kind of schizochytrium limacinums.
Background technique
Docosahexaenoic acid (DHA, C22:6n-3) is the very useful ω -3 race fatty acid of a kind of pair of human health.People
DHA content is very rich in the cerebral cortex of class, retina, testis and sperm, and DHA is that brain structure fatty acid is indispensable
One of ingredient.DHA has very good effect to treatment atherosclerosis, hypertriglyceridemia, hypertension and cancer,
It plays an important role to the development of infant's intelligence and eyesight.Furthermore, it has been reported that DHA has prevention coronary heart disease, heart is reduced
The effects of patient's sudden death probability, prevention senile dementia and anti-aging.The mankind are as lacking needed for synthesis ω -3 race fatty acid
Enzyme, cannot itself synthesis DHA, can only be absorbed from food.Traditionally DHA is extracted from deep sea fish oil, kind, season by fish
Section, the influence in geographical location and it is unstable, and lead to DHA also rich in cholesterol and other unsaturated fat sour components in fish oil
Extraction purification higher cost, yield are also limited.
Schizochytrium limacinum (Schizochytrium sp.) also known as splits pot algae, can synthesize DHA, since its modes of reproduction is to split
It grows, growth cycle is short, and the intracellular advantages such as fatty acid and DHA content height, it is considered to be the production comparatively ideal strain of DHA.Choosing
Educating the stable and high schizochytrium limacinum excellent species of fat content is the key that DHA high yield.
Plant cellulose is a type organic the most abundant in nature.It is estimated that plant passes through photosynthesis every year
The dry matter of generation is up to 1500~200,000,000,000 tons, and being uniquely can ultra-large regenerated material object resource on the earth.In China,
The agricultural crops stalk generated every year has more than 700,000,000 tons, is equivalent to 3.5 hundred million tons of standard coals, but be used for industrial process or burning every year
Plant cellulose resource only account for 2% or so, the overwhelming majority is also unutilized.Recent decades due to fossil resource shortage increasingly
Severe and environmental pollution is got worse, using from a wealth of sources and reproducible plant cellulose as the raw material inverting biological energy and production
Industrial chemical receives the extensive concern of countries in the world.Wherein using biotechnology as the bioconversion of the plant cellulose of core
Have become one of bioenergy exploitation, biomass resources processing and key technology of Green Chemical Engineering Process.
It can produce a large amount of glucose, xylose, arabinose and a small amount of nitrogen source after plant cellulose sour water solution, theoretically can
Enough meet the needs of thalli growth metabolism, if it is possible to cultivate fragmentation as culture medium using reproducible cellulosic hydrolysate
Chytrid, then the cost of schizochytrium limacinum fermentation production DHA will be greatly reduced, and save mass energy, while plant cellulose is become useless
For treasured.But in cellulosic hydrolysate other than cell grows required carbon source and nitrogen source, often there is also by-product such as first
Acid, acetic acid, 5 hydroxymethyl furfural etc. are to the virose substance of cell, and the schizochytrium limacinum of wild type substantially can not be in non-detoxification
It is grown in cellulosic hydrolysate.The conventional resolving ideas and method of this problem are that cellulosic hydrolysate is carried out enzyme and detoxification
Processing, makes it meet the growth requirement of schizochytrium limacinum, but During Detoxification takes time and effort, and secondary pollution is serious.
Strain breeding thereof generallys use some physics and chemical mutagenesis method, such as ultraviolet mutagenesis, ion beam mutagenesis and methyl
Mutagens such as sulfonic acid (EMS), nitrosoguanidine (NTG) etc..The screening technique of conventional mutant strain also has very much, such as
Sudan black B stain method, red tetrazolium discoloration method, the high sugared facture of low temperature, flow cytometer screening method etc..But these methods
There are toxicity big, low efficiency, the non-intuitive disadvantage of screening process.
Therefore, inventor attempts the new method breeding schizochytrium limacinum type bacterium resistant to the cellulosic hydrolysate of non-detoxification
Strain, it is expected that the bacterial strain filtered out can be grown directly upon in the cellulosic hydrolysate without detoxification treatment, and then efficiently to give birth to
Produce DHA.
Summary of the invention
One of the objects of the present invention is to provide a kind of selection of schizochytrium limacinum, this method can breeding be mutated
Type schizochytrium limacinum bacterial strain, it is resistant to the toxicant in bagasse hydrolyzate, it can be in the sugarcane pulp water without detoxification
It solves growth metabolism in liquid and produces DHA.
Another object of the present invention is to provide a kind of schizochytrium limacinums that the bagasse hydrolyzate to non-detoxification is resistant
Mutant strain, the bacterial strain growth metabolism and can produce DHA directly in the bagasse hydrolyzate of non-detoxification.
It is of the invention the technical solution adopted is as follows:
The present invention provides a kind of selection of schizochytrium limacinum, includes the following steps:
(1) bacteria suspension of the initial thallus of schizochytrium limacinum is prepared；
(2) bacteria suspension is coated on bagasse hydrolyzate solid medium and is cultivated；
The bagasse hydrolyzate solid medium includes bagasse hydrolyzate, salinity and agar；The bagasse hydrolysis
The concentration of liquid makes the death rate of thallus be higher than 90%；
The bagasse hydrolyzate is bagasse hydrolysising original liquid or the dilution diluted by the bagasse hydrolysising original liquid
Liquid；The bagasse hydrolysising original liquid is bagasse to be carried out acid heat processing, then adjust what pH to 5-7 was obtained；
(3) single colonie survived on the bagasse hydrolyzate solid medium is selected, respectively secondary culture；
(4) selection can stablize passage and the high bacterial strain of thallus DHA yield.
Selection according to the present invention, the initial thallus of schizochytrium limacinum described in step (1) any can split to be commercially available
Chytrid thallus is grown, the schizochytrium limacinum thallus of high yield DHA is preferably capable.
Selection according to the present invention, it is preferable that step (1) method for preparing schizochytrium limacinum bacteria suspension is: will
The initial thallus of schizochytrium limacinum carries out activation culture, obtains schizochytrium limacinum seed bacterium solution；The schizochytrium limacinum seed bacterium solution is used
Sterile saline dilution, centrifugation, thallus are resuspended using sterile saline, obtain bacteria suspension.The thallus activation culture
Known schizochytrium limacinum activation method in field can be used in method.A kind of preferred embodiment according to the present invention, the thallus
The method of activation culture are as follows: the initial thallus of schizochytrium limacinum is inoculated in Solid media for plates, cultivates 36 at 25~30 DEG C
~48h, obtains single colonie；Single colonie is inoculated in seed fluid nutrient mediums of saccharomycete, 24~36h is cultivated at 25~30 DEG C, obtains institute
State schizochytrium limacinum seed bacterium solution.The Solid media for plates and the seed fluid nutrient mediums of saccharomycete can be used those of known.
Preferred embodiment according to the present invention, the composition of the Solid media for plates are as follows: glucose is 30~60g/L, peptone
It is 5~15g/L for 10~20g/L, yeast extract, sea crystal is 20~30g/L, 15~20g/L of agar.The seed liquid training
Support the composition of base are as follows: glucose is 30~60g/L, peptone is 10~20g/L, yeast extract is 5~15g/L, and sea crystal is
20~30g/L.Using above-mentioned seed fluid nutrient mediums of saccharomycete, schizochytrium limacinum growth rate is fast, and activity is high, is coated on bagasse hydrolysis
It is easier to survive when in liquid solid medium and be proliferated.
Selection according to the present invention, it is preferable that in step (1), the bacteria suspension concentration is 106~108cfu。
Selection according to the present invention, in step (2), the bagasse is that sugarcane squeezes the waste material after sugar.Invention human hair
Existing, the nutritional ingredient of bagasse hydrolyzate is suitble to schizochytrium limacinum to grow and produce DHA, and bagasse abundant raw material, cheap.
Preferably, the bagasse controls water content in 5wt% or less by being dried.A kind of preferred implementation according to the present invention
Mode, the temperature of the drying process are 60~80 DEG C, the time 2~3 days, optionally, while increasing aeration operation, with enhancing
Drying effect.It is dried for example, air dry oven can be used.Selection according to the present invention, it is preferable that described sweet
Bagasse carries out pulverization process before it is dried or after dry, preferably carries out pulverization process after the drying.It is preferred real according to the present invention
Mode is applied, it is 2~20mm that the bagasse powder, which is broken to partial size,.It is highly preferred that the bagasse is sieved after crushing using mesh screen
Get bagasse powder.The aperture of preferred embodiment according to the present invention, the mesh screen is less than 16mm.
Selection according to the present invention, in step (2), the bagasse hydrolysising original liquid is by mixing bagasse and acid solution
It closes, heat treatment, then obtained through adjusting pH；The acid solution can be at least one of sulfuric acid, hydrochloric acid, nitric acid.More preferably
For sulfuric acid.Adjusting pH lye can be sodium hydrate aqueous solution, potassium hydroxide aqueous solution, calcium hydroxide aqueous solution etc., preferably
Calcium hydroxide aqueous solution.A kind of preferred embodiment according to the present invention, the preparation of bagasse hydrolysising original liquid described in step (2)
Method are as follows: bagasse is mixed with acid solution according to solid-to-liquid ratio for 1:2~8,60~200min, mistake are reacted at 100~160 DEG C
Filter is added lye and adjusts pH to 5~7；The hydrogen ion concentration of the acid solution is 0.01~0.05mol/L.It is a kind of according to the present invention
Preferred embodiment, in step (2), salinity is added by bagasse hydrolysising original liquid in the bagasse hydrolyzate solid medium
Be made with agar, the bagasse hydrolysising original liquid the preparation method comprises the following steps: according to solid-to-liquid ratio being 1:3 by bagasse and aqueous sulfuric acid
~6 mixing, react 60~120min at 120~150 DEG C, and filtering is added lye and is adjusted to pH5~7；The concentration of the sulfuric acid
For 0.01~0.02mol/L.
Selection according to the present invention, it is preferable that in step (2), the concentration of the bagasse hydrolyzate makes thallus
The death rate is higher than 95%, more preferably higher than 99%, is further preferably higher than 99.9%.It is to be understood that the death rate cannot be 100%,
Otherwise subsequent breeding step can not be carried out.Inventors have found that schizochytrium limacinum is for non-detoxification and the smaller bagasse of extension rate
Hydrolyzate tolerance is lower, and thallus lethality is high, meanwhile, for that can survive in the smaller bagasse hydrolyzate of extension rate
Schizochytrium limacinum bacterial strain, tolerance is often stronger, also has higher DHA yield；And suitably reduce bagasse hydrolyzate
Concentration, then help to improve the survival rate of schizochytrium limacinum, to increase breeding sample size.Inventors have found that when using bagasse
It is the range that comparison is suitble to strain breeding thereof when 1~2 times of dilution of hydrolysising original liquid.And a kind of preferred implementation according to the present invention
Mode, the bagasse hydrolyzate use bagasse hydrolysising original liquid.
Selection according to the present invention, it is preferable that in step (2), above-mentioned bacteria suspension is taken to be coated on bagasse hydrolyzate
In solid medium, cultivated 3~4 days at 25~30 DEG C.
Selection according to the present invention, it is preferable that in step (2), the salinity is normal according to culture schizochytrium limacinum
Rule formula addition.In general, the cationic constitution element in the salinity includes Na, Mg, K and N, it can also contain Fe, it is described
Anion constitution element in salinity includes S and P.Specific component may include Na2SO4、NaCl、MgSO4、(NH4)2SO4With
KH2PO4, preferably containing microelements such as Fe, B, Zn.The group of a kind of preferred embodiment according to the present invention, the salinity becomes
The Na of 1~5g/L2SO4, 0.5~1g/L MgSO4, 0.25~0.5g/L (NH4)2SO4, 1~3g/L KH2PO4, 10~
The sea crystal of 20g/L.The sea crystal is the product of seawater and salt chemical engineering, mainly by being evaporated to seawater or brine,
A series of processes such as centrifugation, concentration are simultaneously added microelement and produce.Commercially available sea crystal can meet demand.Separately
Outside, the sea crystal also can be used the seawater of 50~100vt% or artificial sea water to replace, and the composition of the artificial sea water includes NaCl
15~18g/L, NaNO30.5~1.0g/L, KCl 0.5~1.0g/L, MgCl20.5~1.0g/L, K2HPO40.3~
0.5g/L, Na2HPO40.3~0.5g/L, HBO30.35g/L, FeCl3·6H2O 0.05~0.10g/L, MnCl2·4H2O 3
~5mg/L, ZnSO4·7H2O 3~5mg/L, CuSO4·5H2O 2~4mg/L, CoCl2·6H20.3~0.5mg/L of O etc..
Selection according to the present invention, it is preferable that secondary culture described in step (3) still uses described in step (2)
Bagasse hydrolyzate solid medium, secondary culture temperature are 25~30 DEG C, cultivation cycle 3~4 days.
Selection according to the present invention, it is preferable that " stablize passage " described in step (4), be preferably able to stablize biography
In generation 5, is more than generation.
Selection according to the present invention, it is preferable that the yield of thallus DHA described in step (4) can be by by the energy
It measures and obtains after enough strain fermentation cultures for stablizing passage.The fermented and cultured uses Liquid Culture, and fluid nutrient medium can be
Known Schizochytrium limacinum fermentation medium；Salinity can also be added by bagasse hydrolyzate, sterilizing is made.The bagasse training
The preparation of nutrient solution and salinity and composition are described above, repeat no more.Preferably, the condition of the fermented and cultured are as follows: temperature is
25~30 DEG C, fermentation period is 80~140h, more preferably 100~120h.It is highly preferred that the inoculum concentration of the fermented and cultured is
0.5~1mL/50ml is cultivated using shaking, and revolving speed is 180~220r/min.
In step (4), thallus DHA yield can measure to obtain according to conventional method in that art.Breeding side according to the present invention
Method, it is preferable that the measuring method of thallus DHA yield is: fermentation liquid obtained by fermented and cultured is centrifuged, it is preferable over 8000~
It is centrifuged under 10000r/min revolving speed, obtains thallus, the thallus brine preferably washs 2~3 times, is divided into two
Part, portion is dry, measures Fungal biodiversity, another calculates DHA for measuring DHA content in thallus fat content and grease
Yield.The calculation method of DHA yield are as follows:
DHA content in DHA yield=Fungal biodiversity × thallus fat content × grease
The drying process is preferable at 80~100 DEG C dry 48~60h.DHA in the thallus fat content and grease
Content is measured using GC-MS method.
A kind of preferred embodiment according to the present invention, the measurement side of DHA content in the thallus fat content and grease
Method includes the following steps:
1. extracting grease: the thallus hydrochloric acid solution of 6~8mol/L is resuspended, 30~90min of acidolysis at 60~80 DEG C,
N-hexane-ethyl alcohol (2~3:1, v/v) is added to extract 2~3 times, merges supernatant, dries up organic solvent, obtains grease；
2. esterification: by the grease, n-hexane, methanol and concentrated hydrochloric acid, grease, n-hexane, methanol, concentrated hydrochloric acid is added
Mass volume ratio be 10mg:2~3mL:1~3mL:0.1~0.3mL, 60~90min of esterification at 60~65 DEG C；Institute
It states concentrated hydrochloric acid and refers to that mass fraction is more than the hydrochloric acid of 37wt%；
3. assay: measuring esterification reaction product using GC-MS method, obtain DHA in thallus fat content and grease and contain
Amount.
A kind of preferred embodiment according to the present invention, GC-MS parameter are as follows: HP~INNOWAX polarity capillary column is used,
0.25μm×250μm×30m；Carrier gas: He；Carrier gas flux: 1.0mL/min, constant flow rate；Loading mode: it shunts, split ratio
20:1；1 μ L of sample volume；280 DEG C of injector temperature；280 DEG C of detector temperature；Temperature program: 150 DEG C of holding 1min, 10 DEG C/
Min is warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.3min, solvent delay time 3min are run after 240 DEG C,
Scanning mode: full scan.
The present invention also provides the schizochytrium limacinum FNU1 obtained by above-mentioned selection.Schizochytrium limacinum FNU1's of the invention
Depositary institution is China typical culture collection center (CCTCC), and depositary institution address is that Wuhan City, Hubei China province Wuhan is big
It learns, the deposit date is on October 23rd, 2015, deposit number was CCTCC NO:M 2015636；Classification naming is schizochytrium limacinum
FNU1(Schizochytrium sp.FNU1).The Fungal biodiversity (i.e. dry cell weight) of the bacterial strain reaches 30.13g/L, utilizes
GC-MS measures thallus fat content and is up to 44.11wt%, and DHA content is 49.24wt% in grease, and DHA yield is 6.58g/
L。
Compared with prior art, the invention has the following advantages that
Selection provided by the invention is simple and fast, and raw material is easy to get, low in cost, substantially increases breeding efficiency.It is first
It is secondary to have filtered out the schizochytrium limacinum bacterial strain resistant to its using the bagasse hydrolyzate of non-detoxification.Fragmentation pot provided by the invention
Bacteria strain can in the bagasse hydrolyzate of non-detoxification growth metabolism, be not required to addition exogenous carbon and nitrogen source, and can be high
Synthetic Oil and DHA, low production cost are imitated, while improving the utility value of agricultural residue bagasse.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, but protection scope of the present invention is not limited to
This.
In the present invention, the extension rate of the dilution is explained as follows: N times of dilution refers to that the volume of dilution is stoste
N times, when N be 1 when, it is actually not diluted, why extension rate can be 1, be entirely in order to express easily.
Unless otherwise specified, experimental method described in following embodiment is conventional method；Material used, reagent
Deng obtaining from commercial channels.Preparation example and sea crystal used in the examples are that Tianjin Hangu is prospered together the production of sea crystal factory.It adopts
Schizochytrium limacinum initial strains are the schizochytrium limacinum (Schizochytrium sp.) that this field circulation uses, can be from market
Upper purchase can also be asked for the scientific research institution (such as Fujian Normal University) for preserving the strain.
Bibliography: T.Yaguchi, S.Tanaka, T.Yokochi, T.Nakahara,
T.Higashihara.Production of high yields of docosahexaenoic acid by
Schizochytriumsp.strain SR21.Journal of the American Oil Chemists'Society.74,
11:1431-1434.(1997)
Preparation example 1
The preparation method of bagasse hydrolyzate, comprising the following steps:
(1) bagasse after squeezing sugar is taken, is placed in air dry oven, it is 3 days dry at 60 DEG C, until its water content is lower than
5wt%；
(2) bagasse after drying is crushed with pulverizer, is sieved, obtain the bagasse powder that partial size is 2~20mm；
(3) by the H of bagasse powder and 0.01mol/L2SO4Solution is 1:6 mixing according to solid-to-liquid ratio, is reacted at 120 DEG C
60min, filtering, filtrate use Ca (OH)2It is adjusted to pH 5, stands 1h, filtering obtains bagasse hydrolysising original liquid.
Preparation example 2
The preparation method of bagasse hydrolyzate, comprising the following steps:
(1) bagasse after squeezing sugar is taken, is placed in air dry oven, it is 2 days dry at 80 DEG C, until its water content is lower than
5wt%；
(2) bagasse after drying is crushed with pulverizer, is sieved, obtains the bagasse powder of 2~16mm of partial size；
(3) by the H of bagasse powder and 0.02mol/L2SO4Solution is 1:3 mixing according to solid-to-liquid ratio, is reacted at 150 DEG C
120min, filtering, filtrate use Ca (OH)2It is adjusted to pH7, stands 1h, filtering obtains bagasse hydrolysising original liquid.
Preparation example 3
The preparation method of bagasse hydrolyzate, comprising the following steps:
(1) bagasse after squeezing sugar is taken, is placed in air dry oven, it is 2.5 days dry at 70 DEG C, until its water content is lower than
5wt%；
(2) bagasse after drying is crushed with pulverizer, is sieved, obtains the bagasse powder of 2~15mm of partial size；
(3) by the H of bagasse powder and 0.01mol/L2SO4Solution is 1:8 mixing according to solid-to-liquid ratio, is reacted at 140 DEG C
200min, filtering, filtrate use Ca (OH)2It is adjusted to pH6, stands 2h, filtering obtains bagasse hydrolysising original liquid.
Preparation example 4
The preparation of bagasse hydrolyzate solid medium:
By the bagasse hydrolysising original liquid of preparation example 1, the Na of 3g/L is added2SO4, 0.75g/L MgSO4, 0.25g/L
(NH4)2SO4, 3g/L KH2PO4, the sea crystal of 20g/L, 20g/L agar, sterilizing obtains bagasse hydrolyzate solid culture
Base；
The preparation of bagasse hydrolyzate fluid nutrient medium:
By the bagasse hydrolysising original liquid of preparation example 1, the Na of 3g/L is added2SO4, 0.75g/L MgSO4, 0.25g/L
(NH4)2SO4, 3g/L KH2PO4, 20g/L sea crystal, sterilizing, obtain bagasse hydrolyzate fluid nutrient medium.
Preparation example 5
The preparation of bagasse hydrolyzate solid medium:
By the bagasse hydrolysising original liquid of preparation example 2,1.1 times are diluted with water, the Na of 3g/L is added2SO4, 0.75g/L
MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, the sea crystal of 20g/L, 20g/L agar, sterilizing obtains bagasse
Hydrolyzate solid medium；
The preparation of bagasse hydrolyzate fluid nutrient medium:
By the bagasse hydrolysising original liquid of preparation example 2,1.1 times are diluted with water, the Na of 3g/L is added2SO4, 0.75g/L
MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, 20g/L sea crystal, sterilizing, obtain bagasse hydrolyzate liquid training
Support base.
Preparation example 6
The preparation of bagasse hydrolyzate solid medium:
By the bagasse hydrolysising original liquid of preparation example 3,1.5 times are diluted with water, the Na of 3g/L is added2SO4, 0.75g/L
MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, the sea crystal of 20g/L, 20g/L agar, sterilizing obtains bagasse
Hydrolyzate solid medium；
The preparation of bagasse hydrolyzate fluid nutrient medium:
By the bagasse hydrolysising original liquid of preparation example 3,1.5 times are diluted with water, the Na of 3g/L is added2SO4, 0.75g/L
MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, 20g/L sea crystal, sterilizing, obtain bagasse hydrolyzate liquid training
Support base.
Preparation example 7
The preparation of bagasse hydrolyzate solid medium:
By the bagasse hydrolysising original liquid of preparation example 1,2 times are diluted with water, the Na of 3g/L is added2SO4, 0.75g/L MgSO4、
(the NH of 0.25g/L4)2SO4, 3g/L KH2PO4, the sea crystal of 20g/L, 20g/L agar, sterilizing obtains bagasse hydrolyzate
Solid medium；
The preparation of bagasse hydrolyzate fluid nutrient medium:
By the bagasse hydrolysising original liquid of preparation example 1,2 times are diluted, the Na of 3g/L is added2SO4, 0.75g/L MgSO4、
(the NH of 0.25g/L4)2SO4, 3g/L KH2PO4, 20g/L sea crystal, sterilizing, obtain bagasse hydrolyzate fluid nutrient medium.
Preparation example 8
The preparation of fermentation medium:
Glucose 90g/L, peptone 15g/L, yeast extract 15g/L are weighed, sea crystal 25g/L, 3g/L's
Na2SO4, 0.75g/L MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, add water constant volume, sterilize, obtain fermented and cultured
Base.
Embodiment 1
The method of breeding schizochytrium limacinum, includes the following steps:
(1) take the schizochytrium limacinum Schizochytrium sp of -80 DEG C of preservations, streak inoculation on Solid media for plates,
48h is activated at 25 DEG C, obtains single colonie；Gained single colonie is inoculated in seed fluid nutrient mediums of saccharomycete, shaking table is trained at 25 DEG C
36h is supported, liquid amount 50mL/250mL, shaking speed 180r/min obtain seed bacterium solution；By gained seed bacterium solution nothing
The dilution of bacterium water, centrifugation operate 2 times, then thallus resuspension is diluted to 10 with sterile saline6~108Cfu obtains bacteria suspension；
Wherein, the formula of Solid media for plates are as follows: glucose 30g/L, peptone 10g/L, yeast extract 5g/
L, sea crystal 25g/L, agar 20g/L；The formula of the seed fluid nutrient mediums of saccharomycete are as follows: glucose 30g/L, peptone are
10g/L, yeast extract 5g/L, sea crystal 25g/L；
(2) bacteria suspension is coated on the bagasse hydrolyzate solid medium of preparation example 4, is cultivated 4 days at 25 DEG C；
(3) single colonie survived on bagasse hydrolyzate solid medium is selected, secondary culture, culture medium still use respectively
The bagasse hydrolyzate solid medium of preparation example 4 is cultivated at 25 DEG C, and the period is 4 days；
(4) selection can stablize the bacterial strain more than generation of passage 5, be placed in the bagasse hydrolyzate fluid nutrient medium of preparation example 4
Middle fermented and cultured, temperature are 25 DEG C, fermentation period 100h, inoculum concentration 1mL/50ml, shaking speed 220r/min；Measurement
The DHA yield of fermentation thalli selects the higher bacterial strain of DHA yield.
Embodiment 2
(1) bacteria suspension is prepared according to the method for 1 step of embodiment (1)；
(2) bacteria suspension is coated on the bagasse hydrolyzate solid medium of preparation example 5, is cultivated 4 days at 25 DEG C；
(3) single colonie survived on bagasse hydrolyzate solid medium is selected, secondary culture, culture medium still use respectively
The bagasse hydrolyzate solid medium of preparation example 5 is cultivated at 25 DEG C, the period 4 days；
(4) selection can stablize the bacterial strain more than generation of passage 5, be placed in the bagasse hydrolyzate fluid nutrient medium of preparation example 5
Middle fermented and cultured, temperature are 25 DEG C, fermentation period 100h, inoculum concentration 1mL/50ml, shaking speed 220r/min；Measurement
The DHA yield of fermentation thalli selects the higher bacterial strain of DHA yield.
Embodiment 3
(1) bacteria suspension is prepared according to the method for 1 step of embodiment (1)；
(2) bacteria suspension is coated on the bagasse hydrolyzate solid medium of preparation example 6, is cultivated 4 days at 25 DEG C；
(3) single colonie survived on bagasse hydrolyzate solid medium is selected, secondary culture, culture medium still use respectively
The bagasse hydrolyzate solid medium of preparation example 6 is cultivated at 25 DEG C, the period 4 days；
(4) selection can stablize the bacterial strain more than generation of passage 5, be placed in the bagasse hydrolyzate fluid nutrient medium of preparation example 6
Middle fermented and cultured, temperature are 25 DEG C, fermentation period 100h, inoculum concentration 1mL/50ml, shaking speed 220r/min；Measurement
The DHA yield of fermentation thalli selects the higher bacterial strain of DHA yield.
Embodiment 4
(1) bacteria suspension is prepared according to the method for 1 step of embodiment (1)；
(2) bacteria suspension is coated on the bagasse hydrolyzate solid medium of preparation example 7, is cultivated 4 days at 25 DEG C；
(3) single colonie survived on bagasse hydrolyzate solid medium is selected, secondary culture, culture medium still use respectively
The bagasse hydrolyzate solid medium of preparation example 7 is cultivated at 25 DEG C, the period 4 days；
(4) selection can stablize the bacterial strain more than generation of passage 5, be placed in fermented and cultured in the fermentation medium of preparation example 8, temperature
Degree is 25 DEG C, fermentation period 100h, inoculum concentration 1mL/50ml, shaking speed 220r/min；Measure the DHA of fermentation thalli
Yield selects the higher bacterial strain of DHA yield.
Test example 1
In Examples 1 to 4, thallus lethality is shown in Table 1 after step (2) culture.
Table 1
| Embodiment | 1 | 2 | 3 | 4 |
| Lethality (%) | 99.9 | 96.8 | 94.2 | 89.5 |
Test example 2
In multiple bacterial strains that embodiment 1-4 breeding obtains, the schizochytrium limacinum bacterial strain of high yield DHA has been obtained
Schizochytrium sp.FNU1, the bacterial strain are screened to obtain by the method for embodiment 1, and inhereditary feature and the performance of DHA yield are prominent
Out, applicant has been preserved in China typical culture collection center (CCTCC), and deposit number is CCTCC NO:M
2015636。
The resistance verification test of mutant strain Schizochytrium sp.FNU1:
(1) mutant strain Schizochytrium sp.FNU1 is inoculated in seed culture fluid, shaking table is trained at 25 DEG C
36h is supported, shaking speed 220r/min, liquid amount 50mL/250mL obtain seed liquor；The seed culture formula of liquid are as follows: 3g/
The Na of L2SO4, 0.75g/L MgSO4, 0.25g/L (NH4)2SO4, 3g/L KH2PO4, 20g/L sea crystal；
(2) after bagasse hydrolysising original liquid prepared by preparation example 1 sterilizing, being cooling, by seed liquor culture transferring obtained by step (1)
In the culture solution, inoculum concentration 1mL/50ml；The shaker fermentation culture at 25 DEG C, shaking speed 220r/min, liquid amount
Fermentation liquid is obtained for 50mL/250mL, fermentation period 120h；
(3) fermentation liquid is centrifuged under 8000r/min revolving speed, obtains thallus, thallus brine 2
Two parts are divided into after secondary, portion dry 60h at 80 DEG C calculates biomass, another is measured for grease, DHA content；
The method for extracting grease are as follows: the thallus hydrochloric acid solution of 6mol/L is resuspended, the acidolysis 30min at 80 DEG C is added
N-hexane-ethyl alcohol (3:1, v/v) extracts 2 times, merges supernatant, dries up organic solvent, obtains grease；
By the grease, n-hexane, methanol and concentrated hydrochloric acid is added, wherein the quality of grease, n-hexane, methanol, concentrated hydrochloric acid
Volume ratio is 10mg:2mL:1mL:0.1mL, and esterification 60min, obtains esterification products at 65 DEG C；
Using DHA content in GC-MS measurement thallus fat content, grease；GC-MS uses Agilent 6890N-5975C,
HP-INNOWAX polarity capillary column, 0.25 μ m, 250 μ m 30m；Carrier gas: He；Carrier gas flux: 1.0mL/min, steady flow
Amount；Loading mode: it shunts, split ratio 20:1；1 μ L of sample volume；280 DEG C of injector temperature；280 DEG C of detector temperature；Heat up journey
Sequence: 150 DEG C of holdings 1min, 10 DEG C/min are warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.It is run after 240 DEG C
3min, solvent delay time 3min, scanning mode: full scan.
Fermentation results are specific as follows:
The Fungal biodiversity (i.e. dry cell weight) of fermentation ends, mutant strain Schizochytrium sp.FNU1 reaches
23.67g/L, measuring fat content using gas chromatograph-mass spectrometer is 23.27%, DHA content 35.70%, calculates DHA yield and is
1.97g/L.
Test example 3
The DHA yield of initial strains Schizochytrium sp. and mutant strain Schizochytrium sp.FNU1 is tried
It tests:
(1) by schizochytrium limacinum initial strains Schizochytrium sp. and mutant strain Schizochytrium
Sp.FNU1 is inoculated in seed culture fluid respectively, the shaking table culture 36h at 25 DEG C, shaking speed 220r/min, and liquid amount is
50mL/250mL obtains seed liquor；The seed culture formula of liquid are as follows: the Na of 3g/L2SO4, 0.75g/L MgSO4、0.25g/L
(NH4)2SO4, 3g/L KH2PO4, 20g/L sea crystal；
(2) after fermentation medium prepared by preparation example 8 sterilizing, being cooling, by seed liquor culture transferring obtained by step (1) in this
In culture solution, inoculum concentration is 1mL/50ml culture solution；The shaker fermentation culture at 25 DEG C, shaking speed 220r/min fill liquid
Amount is 50mL/250mL, and fermentation period 120h obtains fermentation liquid；
(3) fermentation liquid is centrifuged under 8000r/min revolving speed, obtains thallus, the thallus brine
Two parts are divided into after 2 times, portion dry 60h at 80 DEG C calculates biomass, another is measured for grease, DHA content；
The method for extracting grease are as follows: the thallus hydrochloric acid solution of 6mol/L is resuspended, the acidolysis 30min at 80 DEG C is added
N-hexane-ethyl alcohol (3:1, v/v) extracts 2 times, merges supernatant, dries up organic solvent, obtains grease；
By the grease, n-hexane, methanol and concentrated hydrochloric acid is added, wherein the quality of grease, n-hexane, methanol, concentrated hydrochloric acid
Volume ratio is 10mg:2mL:1mL:0.1mL, and esterification 60min, obtains esterification products at 65 DEG C；
Using DHA content in GC-MS measurement thallus fat content, grease；GC-MS uses Agilent 6890N-5975C,
HP-INNOWAX polarity capillary column, 0.25 μ m, 250 μ m 30m；Carrier gas: He；Carrier gas flux: 1.0mL/min, steady flow
Amount；Loading mode: it shunts, split ratio 20:1；1 μ L of sample volume；280 DEG C of injector temperature；280 DEG C of detector temperature；Heat up journey
Sequence: 150 DEG C of holdings 1min, 10 DEG C/min are warming up to 200 DEG C, and 2 DEG C/min is warming up to 220 DEG C, keeps 5min.It is run after 240 DEG C
3min, solvent delay time 3min, scanning mode: full scan.
Fermentation results are specific as follows:
Initial strains Schizochytrium sp. Fungal biodiversity (i.e. dry cell weight) is 30.37g/L, is joined using makings
Measuring fat content with instrument is 33.28%, and DHA content 47.10%, calculating DHA yield is 4.7g/L.
Mutant strain Schizochytrium sp.FNU1 Fungal biodiversity (i.e. dry cell weight) reaches 30.13g/L, utilizes
It is 44.11% that gas chromatograph-mass spectrometer, which measures fat content, DHA content 49.24%, and calculating DHA yield is 6.58g/L.
Present invention is not limited to the embodiments described above, without departing from the essence of the present invention, this field skill
Any deformation, improvement, the replacement that art personnel are contemplated that each fall within the scope of the present invention.
Claims (1)
1. a kind of schizochytrium limacinum (Schizochytrium sp.) FNU1 bacterial strain, which is characterized in that the bacterial strain is preserved in Chinese allusion quotation
Type culture collection, deposit number are CCTCC NO:M 2015636.
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