CN105331670B - Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi - Google Patents
Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi Download PDFInfo
- Publication number
- CN105331670B CN105331670B CN201510892049.7A CN201510892049A CN105331670B CN 105331670 B CN105331670 B CN 105331670B CN 201510892049 A CN201510892049 A CN 201510892049A CN 105331670 B CN105331670 B CN 105331670B
- Authority
- CN
- China
- Prior art keywords
- kou shi
- hidden dinoflagellate
- culture
- algae
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to microbial fermentations, particularly relate to a kind of method for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi.The hidden dinoflagellate of Kou Shi is inoculated into fermentation in the culture medium containing carbon source, nitrogen source and necessary nutrient, fermentation liquid is made；When the hidden dinoflagellate of Kou Shi enters logarithmic growth phase, pot algae will be split and be inoculated into the fermentation liquid of step A and carry out mixed fermentation；The hidden dinoflagellate of Kou Shi is the hidden dinoflagellate algae of Kou Shi that can accumulate polyunsaturated fatty acid, belong to the hidden dinoflagellate category of Kou Shi, and splitting pot algae is that can accumulate polyunsaturated fatty acid and wherein palmitinic acid mass percentage not less than 30% splits pot algae algae.The present invention, which solves the hidden dinoflagellate of existing Kou Shi and splits pot algae and individually ferment, has the problems such as different degrees of technological deficiency, subsequent processing complicated, high production cost, has many advantages, such as process cycle is short, production cost is low, prepared product not need the subsequent processing of complexity that may conform to the requirement of relevant criterion, the quality of grease high.
Description
Technical field
The present invention relates to microbial fermentations, particularly relate to a kind of method for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi.
Background technique
Polyunsaturated fatty acid (PUFA) refers to containing two or more double bonds, and a length of 18-22 carbon of carbochain is former
The straight chain fatty acid of son, it is the important component of cell biological film, is fatty acid necessary to human body.Vital movement is risen
Particularly important effect, such as PUFA have anticancer, prevention cardiovascular and cerebrovascular disease, improve body immunity, anti-agings, resist
The effects of allergy.Wherein docosahexaenoic acid (DHA) has prior physiological function in PUFA.
DHA is the PUFA of ω -3 series.Research finds that DHA is primarily present in cerebral gray matter, it is brain and retina
The main constituents of middle ω -3PUFA, brain and visual development to infant have very important effect.Simultaneously have anticancer,
Anti-inflammatory, prevention senile dementia, prevention cardiovascular and cerebrovascular disease and diabetes and other effects.If pregnant woman or infant development early stage DHA are mended
Deficiency is filled, the learning and memory ability decline of baby is will cause, additionally will affect the capacity of baby.Therefore expert builds
View, pregnant women and infant development early stage should supplement enough DHA.
The source of traditional DHA is fish oil, but because fish oil fishlike smell weight, and fatty acid composition is complicated, DHA content is low, divides
From purification difficult, many reasons containing pollutants such as heavy metals are easy, far from meeting the needs of existing market.Research hair
Existing a variety of marine microalgaes contain a large amount of PUFA, such as the hidden dinoflagellate of Kou Shi (Crypthecodinium cohnii), split pot algae
(Schizochytrium sp.) and my Ken Shi algae (Ulkenia amoeboida), the Ministry of Public Health of China have also ratified in 2010
New resource food can be used as by the DHA algal oil that these three algaes produce and carry out production and sales.
It the hidden dinoflagellate of Kou Shi and splits pot algae and is all cultivated using stainless steel fermentation tank, wherein the hidden dinoflagellate of Kou Shi is in the world
It is used for the microalgae cell of DHA algal oil industrialized production earliest.Fatty acid composition is simple in the hidden dinoflagellate of Kou Shi, long chain fatty acid fraction
Only DHA and palmitic acid content is low, it is at room temperature liquid condition that the oil extracted is of light color, and grease freezing point is low, benefit
In downstream processing and the application in other food.But the speed of growth of the hidden dinoflagellate of Kou Shi is slower, and incubation time is long, and substrate turns
Rate is low, and the surplus of residual sugar is big in waste liquid after culture, and biomass is low, high production cost, causes the price of product high.And
Split the growth of pot algae (Schizochytrium sp.) cell fastly, biomass is easy to accumulate, and to the high conversion rate of substrate, trains after fermentation
It is low to support residual sugar amount in base, is the common algae of nearly 1 year industrialized production DHA.But split fatty acid in pot algae grease produced
High, the crude oil extracted at room temperature of complicated components, especially the palmitic acid content accounting in total fatty acids dinoflagellate hidden ratio Kou Shi
There is crystallization to be precipitated, or be in solid state, be easy to cause wall built-up in food service industry application, float, stir the problems such as uneven, it is right
The direct application of other industry also results in obstacle.
In traditional grease production technique, crude oil all need to carry out the refinement steps such as oil fractionation (or winterization processing) so that
It keeps liquid under given conditions, to meet the requirement of relevant national standard.Oil fractionation is divided into crystallization and separation two is big
Step, first precipitates crystal grease cooling, then carries out the isolated solid-state fat of crystal-liquid and fluid oil.Solid-state fat master
It to participate in constituting by saturated fatty acid (especially palmitinic acid), then liquid is presented since unsaturated fatty acid content improves in fluid oil
Body state.Grease mentions the product of the refined different stage of Crystallization Procedure process, application range and city by different dividing
Field value differs greatly.
In general, the freezing point of fatty acid is determined by the length of carbochain and the quantity of double bond and position, identical carbon chain lengths
Unsaturated fatty acid it is lower than saturated fatty acid freezing point, double key number is more, and (i.e. degree of unsaturation is higher) freezing point is lower.According to
The regulation of the national standard (GB 26400-2011) of food additives DHA grease, the structural state of DHA oil are oily liquids.Mesh
The preceding DHA crude oil high for palmitic acid content, if not being able to satisfy the requirement of national standard without multistage crystallized abstract,
As CN 102746947B patent document in it is also proposed that the side of DHA and saturated fatty acid in pot algae oil is split in a kind of separation, purifying
Method.The step of content of saturated fatty acid is higher in grease winterization treatment process, required winterization is more complicated, and the processing time gets over
It is long.Because winterization technique is initially formed nucleus by crystallisation by cooling, then induces solid rouge that growth is precipitated around it and form agglomerate
So that filtering is precipitated.When the high melting point component contained in the grease is more, the crystal that is not only formed this agglomeration compared with
It is more, and crystals, entrainment of more liquid, the crystal of generation is unstable, filtration difficulty can not even filter sometimes, make the winter
The efficiency of change reduces, it is desirable to reach ideal winterization effect may need repeatedly to be repeated multi-step crystallization operation or it is other compared with
Complicated crystallization processes, therefore step increases and the time extends the increase that will cause energy consumption, this is industrial with energy-saving
It is required that and developing direction run counter to, production cost has also been increased considerably while reducing production efficiency.Therefore how to pass through
Optimization for fermentation technology reduces the content of saturated fatty acid (especially palmitinic acid) in algae oil to simplify winterization technique, is microbial oil
One of the main direction of studying of rouge industry emerging technology development.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, can be effectively reduced
Residual sugar amount in algae oil after the content and culture of saturated fatty acid (especially palmitinic acid) in fermentation liquid has reached reduction life
Cost is produced, the purpose of production efficiency is improved.
Overall technology design of the invention is:
The method for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, comprises the technical steps that:
A, the hidden dinoflagellate of Kou Shi is inoculated into fermentation in the culture medium containing carbon source, nitrogen source and necessary nutrient and hair is made
Zymotic fluid；
B, when the hidden dinoflagellate of Kou Shi enters mid-term or the later period of logarithmic growth phase, fermentation pot algae will be split being inoculated into step A
Mixed fermentation is carried out in liquid；
The hidden dinoflagellate of the Kou Shi is the hidden dinoflagellate algae of Kou Shi that can accumulate polyunsaturated fatty acid, belongs to the hidden dinoflagellate of Kou Shi
Belong to, splitting pot algae is that can accumulate polyunsaturated fatty acid and wherein palmitinic acid mass percentage not less than 30% splits pot algae algae
Kind.
Since the hidden dinoflagellate of growth rate ratio Kou Shi for splitting pot algae is fast, if preferential culture split pot algae can be to the hidden dinoflagellate of Kou Shi
Growth generates inhibiting effect, therefore the present invention preferentially cultivates the hidden dinoflagellate of Kou Shi, then accesses the seed for splitting pot algae, splits the fermentation of pot algae
The residual sugar in fermentation liquid can be rapidly depleted, to effectively reduce cost and shorten fermentation period.
Particular technique of the invention is conceived also:
Carbon source described in culture medium in the present invention is selected from the mixture of glucose or glucose and glycerol.
More preferably technical implementation way is that the carbon source is glucose.
Nitrogen source described in culture medium of the invention contains yeast extract, sodium glutamate, peptone.
Necessary nutrient described in culture medium of the invention includes phosphate, sulfate, sylvite, magnesium salts, molysite, carbon
Hydrochlorate, manganese salt, cobalt salt, zinc salt, sodium salt, boride, vitamin, biotin.
Further to promote fermentation level, shortens fermentation period, optimize fermentation results, preferred technical solution is, described
Step A in the hidden dinoflagellate inoculum concentration of Kou Shi percent by volume be 3%-10%.
To be more conducive to the hidden dinoflagellate of Kou Shi and splitting the growth of pot algae, the content of effective component in promotion fermentation end products,
Improve residual sugar utilization rate in fermentation liquid.Preferred technical solution is, when the hidden dinoflagellate of Kou Shi enters logarithmic growth in the step B
When the later period of phase, pot algae will be split and be inoculated into the fermentation liquid of step A and carry out mixed fermentation.
In microorganism field, the basic experiment technical ability that cell growth curve is this field is made by cellular liquid culture,
It may determine that laundering period, logarithmic phase, stationary phase and the decline phase of cell growth by growth curve；Cell growth curve is also to sentence
The important indicator for determining cell viability is one of the basic parameter of cultivated cytobiology characteristic.It is thus thin from growth curve judgement
The mid log phase of born of the same parents or later period are the basic common sense of this field, and details are not described herein.
To further increase the utilization rate of carbon source in culture medium, while meeting the needs of fast industrialization fermentation, preferably
Technical implementation way is, the terminal of mixed fermentation is that residual sugar is 0g/L or levels off to 0g/L in fermentation liquid in the step B.
Further to promote fermentation level, shortens fermentation period, optimize fermentation results, split pot algae in the step B and connect
The percent by volume of kind amount is 1%-6%.
To obtain more satisfied fermentation results, the fermentation condition of optimization is that step A, the fermentation condition in B is: temperature
20 DEG C -30 DEG C, revolving speed 100rpm-200rpm.
In fermentation industry, shaking flask culture and fermentation tank culture are common liquid fermentation and culture mode in industry, shaking flask training
The dissolved oxygen in the airing efficiency adjusting culture solution of the rotation for passing through bottle swingging machine or shaking table or hunting speed and bottle stopper is supported, and is fermented
Dissolved oxygen in tank is by ventilating and stirring in conjunction with the dissolved oxygen in control fermentation liquid, and the dissolved oxygen in fermentor needs in actual operation
It asks and realizes equivalence replacement, therefore dissolved oxygen demand after can amplifying calculating by Fermentation Engineering principle according to the cultivation results of shaking flask
It is general in shaking flask culture and fermentation tank culture.
Culture medium in step A is fermentation medium, and preferred embodiment is that every liter of fermentation medium includes following matter
The raw material of amount:
Glucose 40g-100g；Sodium glutamate 1g-4g；Yeast extract 1g-4g；Peptone 1g-4g；NaHCO3 0.1g-
0.7g；Nacl 15g-30g；MgSO4·7H2O 2g-8g；Kcl 0.5g-3g；(NH4)2SO40.1g-1g；Cacl2·2H20
0.1g-1g；KH2PO40.1g-1g；H3BO30.01g-0.05g；Na2EDTA 0.01g-0.06g；Mncl2·4H2O 4×10- 3g-10×10-3g；Fecl3·6H2O 1×10-3g-5×10-3g；Zncl2 0.4×10-3g-1×10-3g；Cocl·6H2O
0.1×10-3g-0.5×10-3g；CuSO4·5H2O 0.01×10-3g-0.04×10-3g；VB16mg-15mg；VB125mg-
9mg；Biotin 1mg-2mg；PH=6.5.
It can easily be shown that being commonly used during industrial fermentation to meet the needs of industrialized production and expanding training
Feeding mode prepares fermentation seed liquor used, and the number for expanding culture can flexibly be controlled according to factors such as the scales of fermentation
System, while the preparation of seed used in fermentation process and fermentation medium belongs to prior art scope, applicant is no longer superfluous herein
It states.Preferred technical implementation way is:
Further include that a seed spreads cultivation step before the step A, i.e., will split pot algae and be inoculated in respectively with the hidden dinoflagellate of Kou Shi
Seed liquor is made through culture in seed culture medium, every liter of seed culture medium includes the raw material of following quality:
Glucose 10g-30g；Peptone 1g-2g；Yeast extract 0.5g-3g；Sodium glutamate 0.5g-2g；Nacl 15g-
30g；MgSO4·7H2O 2g-8g；Kcl 0.5g-3g；(NH4)2SO40.1g-1g；Cacl2·2H20 0.1g-1g；KH2PO4
0.1g-1g；PH=6.5.
The preparation process of the seed liquor is as follows:
(1) preparation of pot algae seed liquor is split
1. accessing a ring solid conservation in the 250 milliliters of triangular flasks equipped with 50 milliliters of aseptic seed culture mediums cooled down
Split pot algae, under 20-30 DEG C, 100-200rpm, dark condition culture to cell enter logarithmic growth phase complete for the first time plant
Son activation；
2. the inoculum concentration for being 5%-10% according to percent by volume, is seeded to the seed culture fluid of activation fresh and has gone out
In the seed culture medium of bacterium, culture carries out second of activation to cell entrance under 20-30 DEG C, 100-200rpm, dark condition
Logarithmic growth phase.
(2) preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
1. the inoculum concentration for being 10%-20% according to percent by volume is being cooled down equipped with 50 milliliters of aseptic seed cultures
The hidden dinoflagellate of Kou Shi that fluid preservation is accessed in 250 milliliters of triangular flasks of base, is trained under 20-30 DEG C, 100-200rpm, dark condition
It supports to cell and enters logarithmic growth phase completion first time algae activation；
2. the culture solution of activation, is accessed fresh seed culture by the inoculum concentration for being 10%-20% according to percent by volume
In base, culture to cell enters logarithmic growth phase and completes to activate for the second time under 20-30 DEG C, 100-200rpm, dark condition.
The present invention collects microalgae cell using technology well known within the skill of those ordinarily skilled, including is centrifuged and filters, and receives
Common and well known technology is dry using those skilled in the art for organism after collection, including freeze-drying or vacuum and heating drying
Method, stored after dry or measurement biomass and grease.The grease being previously mentioned includes phosphatide, free fatty acid and fatty acid
Lipid, the lipid of fatty acid include triacylglyceride, diacylglycerol ester, monoacylglycerol ester, phosphatide, sterol rouge etc..This
Inventing extracted DHA not only includes free DHA, further includes that DHA is formed by various lipids.
The method that the present invention extracts grease uses similar compatibility principle, uses the solvent of dissolvable grease.These solvent packets
Include chloroform-methanol, petroleum ether, n-hexane etc., preferably petroleum ether.The ratio of petroleum ether and frustule amount be 50ml petroleum ether/
100mg dry algae powder.Specific extraction step are as follows: after weighing the grinding of 100mg dry algae powder, after being impregnated 4 hours with petroleum ether, be collected by centrifugation
Petroleum ether, by those skilled in the art, common and well known distillation technique removes petroleum ether, obtains grease.In grease
Fatty acid determination method is that the fatty acid in grease is obtained fatty acid methyl ester by turning esterification, and fatty acid methyl ester is used again
SHIMADZU GC2010 gas-chromatography, RTX-2330 chromatogram column analysis.
Substantive distinguishing features obtained by the present invention and significant technological progress are:
1, the present invention according to the hidden dinoflagellate of Kou Shi and splits the growth characteristic of pot algae, and the ingenious hidden dinoflagellate-of Kou Shi that uses splits pot algae
Sequence raise together training mode, formed, can not only be improved obtained using the fatty acid that pot algae is split in Kou Shi hidden dinoflagellate adjustment
Oil quality, and substrate utilization ratio can be increased substantially, the content of residual sugar in fermentation liquid after cultivating is reduced, culture is shortened
Time effectively reduces production cost.
2, using method grease obtained of the invention, palmitinic acid accounts for total rouge for individually splitting the culture of pot algae
The ratio of fat acid reduces, and reduced ratio highest can be increased to the ratio for accounting for total fatty acids not less than 27.07%, DHA, raised
Ratio can reach not less than 27.62%.
3, the content that saturated fatty acid (especially palmitinic acid) in algae oil is reduced by optimization for fermentation technology, can simplify winterization
Technique shortens the winterization time, improves the efficiency of winterization, reduces energy consumption, meets the industrial requirement of energy-saving and developing direction,
And the subsequent refining process of crude oil is enormously simplified, the production cycle of product is effectively shortened, considerably reduces and is produced into
This.
Specific embodiment
The present invention is described further with reference to embodiments, but should not be construed as limitation of the invention, the present invention
Protection scope be subject to the content of claim record, it is any according to the replacement of equivalent technical elements made by specification,
Protection scope of the present invention is not departed from.
Embodiment 1
Split the hidden dinoflagellate ATCC30556 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 10g；Peptone 1g；Yeast extract 0.5g；Sodium glutamate 0.5g；Nacl 20g；MgSO4·7H2O
2g；Kcl 0.5g；(NH4)2SO40.1g；Cacl2·2H20 0.1g；KH2PO40.1g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
Aseptically according to the inoculum concentration of percent by volume 10%, the bacterial strain of fluid preservation is accessed to the dress cooled down
Have in 250 milliliters of triangular flasks of 50 milliliters of aseptic seed culture mediums, under 20 DEG C, 100rpm, dark condition culture to cell into
Enter logarithmic growth phase and carry out first time activation,；Then culture solution is accessed according still further to the inoculum concentration of percent by volume 10% fresh
Aseptic seed culture medium in, under 20 DEG C, 100rpm, dark condition culture carry out second activate after enter logarithm to cell
Growth period can be used as the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in 250 millis equipped with 50 milliliters of aseptic seed culture mediums
It rises in triangular flask.Culture to cell enters logarithmic growth phase and completes to activate for the first time under 20 DEG C, 100rpm, dark condition；So
Afterwards according still further to the inoculum concentration of percent by volume 5%, culture solution is seeded in fresh aseptic seed culture medium, 20 DEG C,
Culture to cell enters logarithmic growth phase and completes to activate for the second time under 100rpm, dark condition, can be used as splitting pot algae seed liquor.
2, it is mixed
Every liter of fermentation medium is made of the raw material of following quality:
Glucose 40g；Peptone 2g；Sodium glutamate 1g；Yeast extract 1g；NaHCO30.1g；Nacl 20g；
MgSO4·7H2O 2g；Kcl 0.5g；(NH4)2SO40.1g；Cacl2·2H20 0.1g；KH2PO40.1g；H3BO30.01g；
Na2EDTA 0.01g；Mncl2·4H2O 4×10-3g；Fecl3·6H2O1×10-3g；Zncl2 0.4×10-3g；Cocl·
6H2O 0.1×10-3g；CuSO4·5H2O 0.01×10-3g；VB16mg；VB125mg；Biotin 1mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 3% after sterilization and cooling
Algae seed liquor is cultivated under 20 DEG C, 150rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 6%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours, until
When remaining sugar concentration is close to 0g/L, frustule is collected, and measures its fatty acid composition (table 1), fatty acid is formed in identical culture
Under the conditions of individually culture split pot algae for control；Residual sugar is after culture with individually the culture hidden dinoflagellate of Kou Shi is pair under the same conditions
According to.Experimental setup three parallel, and experimental result is the average value of three groups of parallel laboratory test results.
Table 1: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 6.84% | 9.45% |
| C16:0 (ratio for accounting for total fatty acids) | 47.32% | 38.41% |
| C22:6 (ratio for accounting for total fatty acids) | 36.54% | 45.26% |
| Residual sugar content (g/L) after culture | 6.79 | 0.18 |
Using the polyculture method in embodiment 1, the ratio that DHA accounts for total fatty acids in resulting grease is improved to 45.26%,
23.86% is improved than control group, the ratio that palmitinic acid accounts for total fatty acids is down to 38.41%, reduces by 18.83% than control group.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 6.79g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.18g/L, and incubation time is short, and the utilization rate of substrate is largely improved compared with individually cultivating hidden dinoflagellate, drop
Low production cost.
Embodiment 2
Split the hidden dinoflagellate ATCC30556 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality: glucose 30g；Yeast extract 3g；Sodium glutamate 2g；Egg
White peptone 1g；Nacl 15g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO4
0.3g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
The inoculum concentration for being aseptically 20% according to percent by volume fills the hidden dinoflagellate access of the Kou Shi of fluid preservation
Have in 250 milliliters of triangular flasks of 50 milliliters of aseptic seed culture mediums, under 30 DEG C, 200rpm, dark condition culture to cell into
Enter logarithmic growth phase to complete to activate for the first time；Then the inoculum concentration for being 20% according still further to percent by volume accesses culture solution fresh
In aseptic seed culture medium, culture to cell enters logarithmic growth phase and completes to carry out second under 30 DEG C, 200rpm, dark condition
Secondary activation can be used as the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in 250 millis equipped with 50 milliliters of aseptic seed culture mediums
It rises in triangular flask.Culture to cell enters logarithmic growth phase and completes to activate for the first time under 30 DEG C, 200rpm, dark condition；So
Afterwards according still further to percent by volume be 10% inoculum concentration, culture solution is seeded in fresh aseptic seed culture medium, 30 DEG C,
Culture to cell enters logarithmic growth phase and completes to carry out second of activation under 200rpm, dark condition, can be used as splitting pot algae
Sub- liquid.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 60g；Yeast extract 3.0g；Sodium glutamate 3.0g；Peptone 2.0g；NaHCO30.2g；Nacl
25g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；H3BO3
0.03g；Na2EDTA 0.03g；MnCl2·4H2O 8.0×10-3g；Fecl3·6H2O 2.0×10-3g；Zncl2 0.6×10- 3g；Cocl·6H2O 0.2×10-3g；CuSO4·5H2O 0.02×10-3g；VB18.0mg；VB128.0mg；Biotin
1.5mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 5% after sterilization and cooling
Algae seed liquor is cultivated under 30 DEG C, 180rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 2.5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 2).Fatty acid is formed in identical training
Support under the conditions of individually culture split pot algae for control, after culture culture in cane sugar content under the same conditions individually culture bandit
The hidden dinoflagellate of family name is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 2: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 8.63% | 10.04% |
| C16:0 (ratio for accounting for total fatty acids) | 48.09% | 40.31% |
| C22:6 (ratio for accounting for total fatty acids) | 34.42% | 38.56% |
| Residual sugar content (g/L) after culture | 7.50 | 0.56 |
Using the polyculture method in embodiment 2, the ratio that DHA accounts for total fatty acids in resulting grease is improved to 38.56%,
The ratio that palmitinic acid accounts for total fatty acids is down to 40.31%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 7.50g/L sucrose in culture medium, and it is mixed mode, residual sugar is 0.56g/ in culture medium after culture
L, and incubation time is short, and the utilization rate of substrate is largely improved compared with individually cultivating hidden dinoflagellate, reduces life
Produce cost.
Embodiment 3
Split the hidden dinoflagellate ATCC30556 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 20g；Yeast extract 2g；Sodium glutamate 1g；Peptone 1g；Nacl 15g；MgSO4·7H2O5g；Kcl
1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
The inoculum concentration for being aseptically 18% according to percent by volume fills the hidden dinoflagellate access of the Kou Shi of fluid preservation
Have in 250 milliliters of triangular flasks of 50 milliliters of aseptic seed culture mediums, under 28 DEG C, 180rpm, dark condition culture to cell into
Enter logarithmic growth phase to complete to activate for the first time；Then the inoculum concentration for being 18% according still further to percent by volume accesses culture solution fresh
In aseptic seed culture medium, culture to cell enters logarithmic growth phase and completes to live for the second time under 28 DEG C, 180rpm, dark condition
Change, can be used as the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in 250 millis equipped with 50 milliliters of aseptic seed culture mediums
It rises in triangular flask.Culture to cell enters logarithmic growth phase and completes to activate for the first time under 30 DEG C, 180rpm, dark condition；So
Afterwards according still further to percent by volume be 6% inoculum concentration, culture solution is seeded in fresh aseptic seed culture medium, 30 DEG C,
Culture to cell enters logarithmic growth phase and completes to activate for the second time under 180rpm, dark condition, can be used as splitting pot algae seed liquor.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 80g；Peptone 1.0g；Yeast extract 4.0g；Sodium glutamate 3.0g；NaHCO30.2g；Nacl
25g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；H3BO3
0.03g；Na2EDTA 0.03g；MnCl2·4H2O 8.0×10-3g；Fecl3·6H2O 2.0×10-3g；Zncl2 0.6×10- 3g；Cocl·6H2O 0.2×10-3g；CuSO4·5H2O 0.02×10-3g；VB18.0mg；VB128.0mg；Biotin
1.5mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 3% after sterilization and cooling
Algae seed liquor is cultivated under 25 DEG C, 150rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 4.5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 3).Fatty acid is formed in identical training
It is individually cultivated under the conditions of feeding and splits pot algae for control, glucose content is cultivated in culture with independent under the same conditions after culture
The hidden dinoflagellate of Kou Shi is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 3: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 6.06% | 8.82% |
| C16:0 (ratio for accounting for total fatty acids) | 40.29% | 33.55% |
| C22:6 (ratio for accounting for total fatty acids) | 39.89% | 47.28% |
| Residual sugar content (g/L) after culture | 9.07 | 0.15 |
Using the polyculture method in embodiment 3, the ratio that DHA accounts for total fatty acids in resulting grease is improved to 47.28%,
18.53% is improved than control group, the ratio that palmitinic acid accounts for total fatty acids is down to 33.55%, reduces by 16.73% than control group.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 9.07g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.15g/L, and incubation time is short, and the utilization rate of substrate is largely improved compared with individually cultivating hidden dinoflagellate, drop
Low production cost.
Embodiment 4
Split the hidden dinoflagellate ATCC30556 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 30g；Yeast extract 1g；Sodium glutamate 1g；Peptone 2g；Nacl 15g；MgSO4·7H2O8g；Kcl
3g；(NH4)2SO41g；Cacl2·2H20 1g；KH2PO41g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
The inoculum concentration for being aseptically 15% according to percent by volume fills the hidden dinoflagellate access of the Kou Shi of fluid preservation
Have in 250 milliliters of triangular flasks of 50 milliliters of aseptic seed culture mediums, under 25 DEG C, 150rpm, dark condition culture to cell into
Enter logarithmic growth phase to complete to activate for the first time；Then the inoculum concentration for being 15% according still further to percent by volume accesses culture solution fresh
Aseptic seed culture medium in, culture to cell enters logarithmic growth phase and completes second under 25 DEG C, 150rpm, dark condition
Activation, is made the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in 250 millis equipped with 50 milliliters of aseptic seed culture mediums
It rises in triangular flask.Culture to cell enters logarithmic growth phase and completes to activate for the first time under 25 DEG C, 150rpm, dark condition；So
First culture solution is seeded in fresh aseptic seed culture medium by the inoculum concentration for being afterwards 7% according still further to percent by volume, 25
DEG C, culture to cell enters logarithmic growth phase and completes second and activates under 150rpm, dark condition, be made and split pot algae seed liquor.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 80g；Sodium glutamate 4g；Yeast extract 4g；Peptone 1g；NaHCO30.7g；Nacl15g；MgSO4·
7H2O 8.0g；Kcl 3g；(NH4)2SO41g；Cacl2·2H20 1g；KH2PO41g；H3BO30.05g；Na2EDTA 0.06g；
Mncl2·4H2O 10×10-3g；Fecl3·6H2O 5×10-3g；ZnCl2 1.0×10-3g；Cocl·6H2O 0.5×10-3g；
CuSO4·5H2O 0.04×10-3g；VB115mg；VB129mg；Biotin 2mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 3% after sterilization and cooling
Algae seed liquor is cultivated under 20 DEG C, 100rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 4.5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 4).Fatty acid is formed in identical training
Individually culture splits pot algae for control under the conditions of supporting；Residual sugar is after culture with individually the culture hidden dinoflagellate of Kou Shi is under the same conditions
Control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 4: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
According to the method for embodiment 4, splits pot algae and the hidden dinoflagellate mixed culture of Kou Shi is grease obtained, DHA accounts for total fatty acids
Ratio is improved to 48.51%, improves 24.32% than control, and the ratio that palmitinic acid accounts for total fatty acids is down to 34.76%, than control
Reduce by 15.53%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 9.14g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.10g/L, and incubation time is short, and mixed culture mode largely improves compared with the individually culture hidden dinoflagellate of Kou Shi
The utilization rate of substrate, reduces production cost.
Embodiment 5:
Split the hidden dinoflagellate ATCC30772 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 15g；Yeast extract 1.0g；Sodium glutamate 1.0g；Peptone 1g；Nacl 30g；MgSO4·7H2O
3g；Kcl 1.5g；(NH4)2SO40.5g；Cacl2·2H20 0.5g；KH2PO40.5g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
The inoculum concentration for being aseptically 10% according to percent by volume fills the hidden dinoflagellate access of the Kou Shi of fluid preservation
Have in 250 milliliters of triangular flasks of 50 milliliters of aseptic seed culture mediums, under 25 DEG C, 150rpm, dark condition culture to cell into
Enter logarithmic growth phase to complete to activate for the first time；Then the inoculum concentration for being 10% according still further to percent by volume accesses culture solution fresh
Aseptic seed culture medium in, culture to cell enters logarithmic growth phase and completes second under 25 DEG C, 150rpm, dark condition
Activation, is made the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in 250 millis equipped with 50 milliliters of aseptic seed culture mediums
It rises in triangular flask.Culture to cell enters logarithmic growth phase and completes to activate for the first time under 25 DEG C, 150rpm, dark condition；So
Afterwards according still further to percent by volume be 5% inoculum concentration, culture solution is seeded in fresh aseptic seed culture medium, 25 DEG C,
Culture to cell enters logarithmic growth phase and completes to activate for the second time under 150rpm, dark condition, can be used as splitting pot algae seed liquor.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 100g；Sodium glutamate 4g；Yeast extract 4g；Peptone 4g；NaHCO30.4g；Nacl30g；
MgSO4·7H2O 3g；Kcl 1.5g；(NH4)2SO40.5g；Cacl2·2H20 0.5g；KH2PO40.5g；H3BO30.04g；
Na2EDTA 0.05g；Mncl2·4H2O 6×10-3g；Fecl3·6H2O3×10-3g；Zncl2 0.9×10-3g；Cocl·
6H2O 0.3×10-3g；CuSO4·5H2O 0.03×10-3g；VB112mg；VB126mg；Biotin 1.5mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 7% after sterilization and cooling
Algae seed liquor is cultivated under 25 DEG C, 200rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours, until
When remaining sugar concentration is close to 0g/L, frustule is collected, and measures its fatty acid composition (table 5).Fatty acid is formed in identical culture
Under the conditions of individually culture split pot algae for control, after culture in culture medium glucose content under the same conditions individually culture
The hidden dinoflagellate of Kou Shi is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 5: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 8.11% | 9.01% |
| C16:0 (ratio for accounting for total fatty acids) | 43.51% | 34.68% |
| C22:6 (ratio for accounting for total fatty acids) | 37.08% | 46.61% |
| Glucose content (g/L) after culture | 8.99 | 0.09 |
According to the method for embodiment 5, splits pot algae and the hidden dinoflagellate of Kou Shi is mixed obtained grease, DHA accounts for total fat
The ratio of acid is improved to 46.61%, improves 25.7% than control.The ratio that palmitinic acid accounts for total fatty acids is down to 34.68%, compares
According to reduction by 20.29%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experimental result discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, training
After supporting, still residue 8.99g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.09g/L, and incubation time is short, and mixed culture mode largely improves compared with the individually culture hidden dinoflagellate of Kou Shi
The utilization rate of substrate, reduces production cost.
Embodiment 6
Split the hidden dinoflagellate ATCC30556 mixed culture production docosahexaenoic acid of pot algae ATCC 28209 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 25g；Yeast extract 1.5g；Sodium glutamate 1.5g；Peptone 1g；Nacl 25g；MgSO4·7H2O
5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；PH=6.5.Wherein the hidden dinoflagellate of Kou Shi and
The specific preparation incubation of pot algae seed liquor is split with embodiment 5.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 80g；Sodium glutamate 1.5g；Yeast extract 2.5g；Peptone 2.5g；NaHCO30.6g；Nacl
25g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；H3BO3
0.03g；Na2EDTA 0.03g；MnCl2·4H2O 8.0×10-3g；Fecl3·6H2O 2.0×10-3g；Zncl2 0.6×10- 3g；Cocl·6H2O 0.2×10-3g；CuSO4·5H2O 0.02×10-3g；VB18.0mg；VB128.0mg；Biotin
1.5mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 5% after sterilization and cooling
Algae seed liquor is cultivated under 25 DEG C, 150rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 2.5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 6).Fatty acid is formed in identical training
Individually culture splits pot algae for control under the conditions of feeding, and glucose content is trained in culture medium with independent under the same conditions after culture
Supporting the hidden dinoflagellate of Kou Shi is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 6: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 7.97% | 10.01% |
| C16:0 (ratio for accounting for total fatty acids) | 40.11% | 33.21% |
| C22:6 (ratio for accounting for total fatty acids) | 38.01% | 45.61% |
| Glucose content (g/L) after culture | 8.75 | 0.15 |
According to the method for embodiment 6, splits pot algae and the hidden dinoflagellate of Kou Shi is mixed obtained grease, DHA accounts for total fat
The ratio of acid is improved to 45.61%, is improved than control nearly by 20%.The ratio that palmitinic acid accounts for total fatty acids is down to 33.21%, compares
17% is reduced according to group.
Individually the culture hidden dinoflagellate of Kou Shi, experimental result find that the hidden dinoflagellate speed of growth of Kou Shi is slow under the same conditions, training
After supporting, still residue 8.75g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.15g/L, and incubation time is short, and mixed culture largely improves the benefit of substrate compared with individually cultivating hidden dinoflagellate
With rate, production cost is reduced.
Embodiment 7
Split the hidden dinoflagellate ATCC30772 mixed culture production docosahexaenoic acid of pot algae CGMCC No.28209 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 20g；Yeast extract 2g；Sodium glutamate 2g；Peptone 1g；Nacl 25g；MgSO4·7H2O7.0g；
Kcl 2.0g；(NH4)2SO40.7g；Cacl2·2H20 0.7g；KH2PO40.7g；PH=6.5.Wherein the hidden dinoflagellate of Kou Shi and split
The specific culture preparation process of pot algae seed liquor is the same as embodiment 5.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 70g；Sodium glutamate 1g；Yeast extract 2g；Peptone 1.5g；NaHCO30.2g；Nacl 25g；
MgSO4·7H2O 6.0g；Kcl 2.0g；(NH4)2SO40.7g；Cacl2·2H20 0.7g；KH2PO40.7g；H3BO3
0.02g；Na2EDTA 0.04g；MnCl2·4H2O 7×10-3g；FeCl3·6H2O4×10-3g；Zncl2 0.7×10-3g；
Cocl·6H2O 0.4×10-3g；CuSO4·5H2O 0.02×10-3g；VB110.0mg；VB127.0mg；Biotin 1.0mg；
PH=6.5.
The ratio access Kou Shi that fermentation medium is aseptically after sterilization and cooling 10% according to percent by volume is hidden
Dinoflagellate seed liquor is cultivated under 25 DEG C, 150rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to
Pot algae seed liquor is split in the ratio access that percent by volume is 1%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 7).Fatty acid is formed in identical training
Individually culture splits pot algae for control under the conditions of feeding, and glucose content is trained in culture medium with independent under the same conditions after culture
Supporting the hidden dinoflagellate of Kou Shi is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 7: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
According to the method for embodiment 7, splits pot algae and the hidden dinoflagellate of Kou Shi is mixed obtained grease, DHA accounts for total fat
The ratio of acid is improved to 52.21%, improves 27.62% than control.The content that palmitinic acid accounts for total fatty acids is down to 28.45%, than
Control reduces by 27.07%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 8.46g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.21g/L, and incubation time is short, and mixed culture mode largely improves compared with the individually culture hidden dinoflagellate of Kou Shi
The utilization rate of substrate, reduces production cost.
Embodiment 8
Split the hidden dinoflagellate ATCC30772 mixed culture production docosahexaenoic acid of pot algae ATCC28209 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 25g；Yeast extract 1.5g；Sodium glutamate 1.5g；Peptone 1g；Nacl 25g；MgSO4·7H2O
5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；PH=6.5.Wherein the hidden dinoflagellate of Kou Shi and
The specific culture preparation process of pot algae seed liquor is split with embodiment 5.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 50g；Sodium glutamate 1.5g；Yeast extract 2.5g；Peptone 0.5g；NaHCO30.6g；Nacl
25g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；H3BO3
0.03g；Na2EDTA 0.03g；Mncl2·4H2O 8.0×10-3g；Fecl3·6H2O 2.0×10-3g；Zncl2 0.6×10- 3g；Cocl·6H2O 0.2×10-3g；CuSO4·5H2O 0.02×10-3g；VB18.0mg；VB128.0mg；Biotin
1.5mg；PH=6.5.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 5% after sterilization and cooling
Algae seed liquor is cultivated under 25 DEG C, 130rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 3%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 6 hours, until
When remaining sugar concentration is close to 0g/L, frustule is collected, and measures its fatty acid composition (table 8).Fatty acid is formed in identical culture
Under the conditions of individually culture split pot algae for control, after culture culture in glucose content under the same conditions individually culture bandit
The hidden dinoflagellate of family name is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 8: pot algae and the hidden dinoflagellate mixed culture primary fat acid analysis result of Kou Shi are split
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 5.97% | 8.99% |
| C16:0 (ratio for accounting for total fatty acids) | 38.13% | 29.49% |
| C22:6 (ratio for accounting for total fatty acids) | 40.56% | 48.59% |
| Glucose content (g/L) after culture | 6.45 | 0.07 |
According to the method for embodiment 8, splits pot algae and the hidden dinoflagellate of Kou Shi is mixed obtained grease, DHA accounts for total fat
The ratio of acid is improved to 48.59%, improves 19.79% than control.The content that palmitinic acid accounts for total fatty acids is down to 29.49%, than
Control reduces by 22.66%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 6.45g/L glucose in culture medium, and it is mixed mode, residual sugar is in culture medium after culture
0.07g/L, and incubation time is short, and mixed culture largely improves substrate compared with the individually culture hidden dinoflagellate of Kou Shi
Utilization rate, reduce production cost.
Embodiment 9
Split the hidden dinoflagellate ATCC30772 mixed culture production docosahexaenoic acid of pot algae CGMCC No.11382 and Kou Shi
1, the preparation of seed liquor
Every liter of seed culture medium includes the raw material of following quality:
Glucose 20g；Yeast extract 2g；Sodium glutamate 1g；Peptone 1g；Nacl 15g；MgSO4·7H2O5g；Kcl
1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；PH=6.5.
The preparation of the sub- liquid of Kou Shi Crypthecodinium cohnii
The hidden dinoflagellate of the Kou Shi of fluid preservation is accessed nothing by the inoculum concentration for being aseptically 12% according to percent by volume
In bacterium seed culture medium, progress first time activation in 42 hours is cultivated under 22 DEG C, 120rpm, dark condition；Then according still further to body
The inoculum concentration that product percentage is 12% accesses culture solution in fresh sterile seed culture medium, in 22 DEG C, 120rpm, dark condition
Lower culture carries out second of activation and enters logarithmic growth phase to cell, can be used as the sub- liquid of Kou Shi Crypthecodinium cohnii.
Split the preparation of pot algae seed liquor
That aseptically accesses a ring solid conservation splits pot algae in aseptic seed culture medium.30 DEG C, 120rpm,
Progress first time activation in 30 hours is cultivated under dark condition；Then the inoculum concentration for being 7% according still further to percent by volume, by culture solution
It is seeded in fresh aseptic seed culture medium, culture carries out second of activation to cell under 30 DEG C, 120rpm, dark condition
Into logarithmic growth phase, it can be used as and split pot algae seed liquor.
2, it is mixed
Every liter of fermentation medium includes the raw material of following quality:
Glucose 40g；Glycerol 20g；Yeast extract 2.0g；Sodium glutamate 2.0g；Peptone 2.0g；NaHCO30.2g；
Nacl 25g；MgSO4·7H2O 5g；Kcl 1g；(NH4)2SO40.2g；Cacl2·2H20 0.3g；KH2PO40.3g；H3BO3
0.03g；Na2EDTA 0.03g；Mncl2·4H2O 8.0×10-3g；Fecl3·6H2O 2.0×10-3g；Zncl2 0.6×10- 3g；Cocl·6H2O 0.2×10-3g；CuSO4·5H2O0.02×10-3g；VB18.0mg；VB128.0mg；Biotin 1.5mg；
PH=3-12.
Fermentation medium aseptically accesses the hidden first of Kou Shi according to the ratio that percent by volume is 5% after sterilization and cooling
Algae seed liquor is cultivated under 25 DEG C, 150rpm, dark condition, when Kou Shi hidden dinoflagellate is when growing into late log phase, according to body
Pot algae seed liquor is split in the ratio access that product percentage is 3.5%, after splitting the access of pot algae seed liquor, every the residual sugar of survey in 4 hours,
When to remaining sugar concentration close to 0g/L, frustule is collected, and measures its fatty acid composition (table 9).Fatty acid is formed in identical training
Support under the conditions of individually culture split pot algae for control, after culture culture in residual sugar content under the same conditions individually culture bandit
The hidden dinoflagellate of family name is control.Experimental setup three parallel, and experimental result is three parallel average values.
Table 9 splits pot algae and hidden dinoflagellate mixed culture fatty acid composition
| Control | Mixed culture | |
| C14:0 (ratio for accounting for total fatty acids) | 8.46% | 11.22% |
| C16:0 (ratio for accounting for total fatty acids) | 48.85% | 40.44% |
| C22:6 (ratio for accounting for total fatty acids) | 35.04% | 40.23% |
| Residual sugar content (g/L) after culture | 10.24 | 0.58 |
According to the method for embodiment 9, splits pot algae and the hidden dinoflagellate of Kou Shi is mixed obtained grease, DHA accounts for total fat
The ratio of acid is improved to 42.23%, improves 14.81% than control.The content that palmitinic acid accounts for total fatty acids is down to 40.44%, than
Control reduces by 17.22%.
The hidden dinoflagellate of Kou Shi is individually cultivated in experiment discovery under the same conditions, and the hidden dinoflagellate speed of growth of Kou Shi is slow, culture knot
Shu Hou, still residue 10.24g/L sugar in culture medium, and it is mixed mode, residual sugar is 0.58g/L in culture medium after culture,
And incubation time is short, and mixed culture largely improves the utilization of substrate compared with the individually culture hidden dinoflagellate of Kou Shi
Rate reduces production cost.
Claims (9)
1. splitting the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that comprise the technical steps that:
A, the hidden dinoflagellate of Kou Shi is inoculated into fermentation in the culture medium containing carbon source, nitrogen source and necessary nutrient and fermentation is made
Liquid, the percent by volume of the hidden dinoflagellate inoculum concentration of Kou Shi are 3%-10%；
B, when the hidden dinoflagellate of Kou Shi enters mid-term or the later period of logarithmic growth phase, pot algae will be split and be inoculated into the fermentation liquid of step A
Mixed fermentation is carried out, the percent by volume for splitting pot algae inoculum concentration is 1%-6%；
The hidden dinoflagellate of the Kou Shi is the hidden dinoflagellate algae of Kou Shi that can accumulate polyunsaturated fatty acid, belongs to the hidden dinoflagellate category of Kou Shi,
Splitting pot algae is that can accumulate polyunsaturated fatty acid and wherein palmitinic acid mass percentage not less than 30% splits pot algae algae；
Carbon source in the step A is selected from the mixture of glucose or glucose and glycerol；Nitrogen source contains yeast extract, paddy ammonia
Sour sodium, peptone；Inorganic nutritive element includes phosphate, sulfate, sylvite, magnesium salts, molysite, carbonate, manganese salt, cobalt salt, zinc
Salt, sodium salt, boride.
2. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the step A
In carbon source be selected from glucose.
3. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the step B
In enter the logarithmic growth later period when the hidden dinoflagellate of Kou Shi, pot algae will be split and is inoculated into the fermentation liquid of step A and carry out mixed fermentation.
4. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the step B
The terminal of middle mixed fermentation is that residual sugar is 0g/L or levels off to 0g/L in fermentation liquid.
5. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that step A, in B
Fermentation condition be: 20 DEG C -30 DEG C of temperature, revolving speed 100rpm-200rpm.
6. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the step
Culture medium in rapid A is fermentation medium, and every liter of fermentation medium includes the raw material of following quality:
Glucose 40g-100g；Sodium glutamate 1g-4g；Yeast extract 1g-4g；Peptone 1g-4g；NaHCO30.1g-0.7g；
NaC l 15g-30g；MgSO4·7H2O 2g-8g；KC l 0.5g-3g；(NH4)2SO40.1g-1g；C aC l2·2H20
0.1g-1g；KH2PO40.1g-1g；H3BO30.01g-0.05g；Na2EDTA 0.01g-0.06g；MnC l2·4H2O 4×10-3g-10×10-3g；FeC l3·6H2O 1×10-3g-5 ×10-3g；ZnC l2 0.4×10-3g-1×10-3g；CoC l·
6H2O 0.1×10-3g-0.5×10-3g；CuSO4·5H2O 0.01×10-3g-0.04×10-3g；VB16mg-15mg；
VB125mg-9mg；Biotin 1mg-2mg；PH=6.5.
7. the method according to claim 1 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the step
Further include that seed spreads cultivation step before rapid A, i.e., will split pot algae and be inoculated in seed culture medium respectively with the hidden dinoflagellate of Kou Shi through cultivating
Seed liquor is made, every liter of seed culture medium includes the raw material of following quality:
Glucose 10g-30g；Peptone 1g-2g；Yeast extract 0.5g-3g；Sodium glutamate 0.5g-2g；NaC l 15g-
30g；MgSO4·7H2O 2g-8g；KCl 0.5g-3g；(NH4)2SO40.1g-1g；CaC l2·2H200.1g-1g；KH2PO4
0.1g-1g；PH=6.5.
8. the method according to claim 7 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that described splits
The preparation process of pot algae seed liquor is as follows:
That a ring solid conservation is accessed in the 250 milliliters of triangular flasks equipped with 50 milliliters of aseptic seed culture mediums cooled down splits pot
Algae, culture to cell enters logarithmic growth phase completion first time seed activation under 20-30 DEG C, 100-200rpm, dark condition；
The seed culture fluid of activation is seeded to fresh and sterilized seed and trained by the inoculum concentration for being 5%-10% according to percent by volume
It supports in base, culture to cell enters logarithmic growth phase and completes to activate for the second time under 20-30 DEG C, 100-200rpm, dark condition.
9. the method according to claim 7 for splitting pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi, it is characterised in that the bandit
The preparation process of the sub- liquid of family name's Crypthecodinium cohnii is as follows:
The inoculum concentration for being 10%-20% according to percent by volume, in 250 equipped with 50 milliliters of aseptic seed culture mediums cooled down
The hidden dinoflagellate of Kou Shi that fluid preservation is accessed in milliliter triangular flask, is cultivated under 20-30 DEG C, 100-200rpm, dark condition to thin
Born of the same parents enter logarithmic growth phase and complete the activation of first time algae；The inoculum concentration for being 10%-20% according to percent by volume, by activation
Culture solution accesses in fresh seed culture medium, is cultivated under 20-30 DEG C, 100-200rpm, dark condition to cell entrance pair
Number growth period completes second and activates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510892049.7A CN105331670B (en) | 2015-12-04 | 2015-12-04 | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510892049.7A CN105331670B (en) | 2015-12-04 | 2015-12-04 | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105331670A CN105331670A (en) | 2016-02-17 |
| CN105331670B true CN105331670B (en) | 2018-12-04 |
Family
ID=55282433
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510892049.7A Active CN105331670B (en) | 2015-12-04 | 2015-12-04 | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105331670B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161576B (en) * | 2018-09-26 | 2020-08-07 | 武汉中科光谷绿色生物技术有限公司 | Method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013834A (en) * | 2011-09-20 | 2013-04-03 | 中国科学院大连化学物理研究所 | Oil production microbe culture method |
| CN103103130A (en) * | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Production method for lipid through mixed culture of microbes |
-
2015
- 2015-12-04 CN CN201510892049.7A patent/CN105331670B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013834A (en) * | 2011-09-20 | 2013-04-03 | 中国科学院大连化学物理研究所 | Oil production microbe culture method |
| CN103103130A (en) * | 2011-11-10 | 2013-05-15 | 中国石油化工股份有限公司 | Production method for lipid through mixed culture of microbes |
Non-Patent Citations (2)
| Title |
|---|
| 二十二碳六烯酸(DHA)发酵工艺的研究;张琼;《中国优秀硕士学位论文全文数据库工程科技Ⅰ辑》;20150115(第1期);B018-59 * |
| 氧对裂壶藻利用甘油产DHA影响机制及其高密度发酵控制策略的研究;常桂芳;《中国博士学位论文全文数据库基础科学辑》;20140515(第5期);第7-10页第1.3.1和1.3.2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105331670A (en) | 2016-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10188596B2 (en) | Omega-7 fatty acid composition, methods of cultivation of tribonema for production of composition and application of composition | |
| CN105018539B (en) | A method of culture schizochytrium limacinum high yield DHA | |
| Cho et al. | Effects of pH control and concentration on microbial oil production from Chlorella vulgaris cultivated in the effluent of a low-cost organic waste fermentation system producing volatile fatty acids | |
| CN109777607B (en) | Method for purifying DHA (docosahexaenoic acid) crude oil | |
| CA2837215C (en) | Algal lipid compositions and methods of preparing and utilizing the same | |
| CN101892160B (en) | Schizochytrium LX0809 (marine fungus) and industrial application thereof | |
| CN110846346B (en) | Microbial oil rich in Sn-2 DHA, and preparation method and application thereof | |
| CN1986822A (en) | Crypthecodinium connii fermenting process for producing docosahexaenoic acid grease | |
| Wang et al. | Oil crop biomass residue-based media for enhanced algal lipid production | |
| CN108707630B (en) | Regulation and control method for improving EPA content in schizochytrium and application | |
| CN106987528A (en) | One plant production docosahexaenoic acid bacterium and its application | |
| CN104860756A (en) | Cordyceps militaris fermentation liquid culture medium, preparation method thereof and application thereof | |
| CN104655785B (en) | A kind of method analyzing microbial grease composition | |
| CN103451247B (en) | A kind of arachidonic preparation method | |
| CN103642860A (en) | Grease rich in DHA (docosahexenoic acid) and DPA (docosapentaenoic acid) as well as preparation and application of grease | |
| CN105331670B (en) | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi | |
| CN103468575B (en) | Crypthecodinium cohnii mutant strain with high yield of DHA (docosahexaenoic acid), as well as fermentation method and application thereof | |
| CN109266698B (en) | Method for adjusting fatty acid composition components in Mortierella microbial oil | |
| CN114032259B (en) | High-density fermentation and hexadecenoic acid extraction method of saccharomycetes | |
| CN105189769A (en) | Production of omega-3 fatty acids from pythium species | |
| CN106318984B (en) | Method for preparing fatty acid glyceride by oleaginous microorganisms, glyceride and application thereof | |
| CN105400836A (en) | Production method for increasing unsaturated fatty acid content | |
| Takagi et al. | Efficient pH and dissolved CO2 conditions for indoor and outdoor cultures of green alga Parachlorella | |
| CN102586351B (en) | Method for promoting high-efficiency accumulation of arachidonic acid grease | |
| CN114807256B (en) | Application of sodium alginate and ferulic acid in improving EPA yield of oleaginous microorganisms and preparation method of microbial grease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 515041 Guangdong province Shantou Changping Road 90, Suning Plaza Building 2 (8-23), 2106, 2107, 2105, 2108, 2109. Applicant after: Guangdong branch of biological engineering Limited by Share Ltd Address before: 515041 Guangdong Province, Shantou city Longhu District East Road Longsheng industrial zone Guangdong runke Biological Engineering Co. Ltd. Applicant before: Guangdong Runke Bioengineering Co., Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |