CN104031843B - A kind of schizochytrium limacinum and application thereof - Google Patents
A kind of schizochytrium limacinum and application thereof Download PDFInfo
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Abstract
The present invention relates to microbial fermentation engineering field, be specifically related to a kind of schizochytrium limacinum and application thereof.Schizochytrium limacinum, does is its Classification And Nomenclature schizochytrium limacinum (Aurantiochytrium? sp) SD006, the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City is preserved on October 21st, 2013, does is its deposit number CGMCC? No:8383.Described schizochytrium limacinum is applied to be prepared in docosahexenoic acid.Obtained strains fast growth of the present invention, fermentation time is short.Schizochytrium limacinum bacterial strain high density fermentation is utilized to produce docosahexenoic acid.Under above-mentioned fermentation condition, within 60-70 hour, maximum production can be obtained.After 70 hours fermentation, obtain biomass be 180-200g/L, DHA output is 50-65g/L.
Description
Technical field
The present invention relates to microbial fermentation engineering field, be specifically related to a kind of schizochytrium limacinum and application thereof.
Background technology
Docosahexenoic acid (DocosahexaenoicAcid, DHA) is a kind of polyunsaturated fatty acid of needed by human, must supplement from meals.DHA is that central nervous system is unified amphiblestroid important feature fat composition, has brain tonic, improves the function of memory and eyesight, especially can promote fetus and the growth of infant brain cell development, improve teenager's memory, prevent and treat senile dementia etc.Traditionally, DHA is mainly derived from fish oil, but owing to being subject to the impact of overfishing, environmental pollution, seasonal variation etc. very large, being utilized fermentable production gradually and replacing.In present stage, it is a kind of marine microorganism that industrial fermentation produces the topmost bacterial classification of DHA---schizochytrium limacinum (Aurantiochytriumsp.), in its total fatty acids, DHA content is up to 40%-60%.The DHA algal oil utilizing schizochytrium limacinum to produce in 2010 is approved as new resource food by the Ministry of Health.But the Main Bottleneck limiting its further genralrlization is at present that the DHA output in unit cost is lower.
Domestic schizochytrium limacinum bacterial screening and the fermentation of relating to mainly contains 20 sections for the patent of DHA grease at present, is mainly divided into three aspects: the seed selection of (1) excellent species.As patent CN1264967C, CN101892160B, CN103602591A, CN101575584B, CN102199541B, CN102899254A, CN102888348A and CN102864111B, these patents acquire biomass by the schizochytrium limacinum bacterial strain screening, cultivate high growth rates and high DHA content and are up to 120-130g/L, and DHA output is up to 25-35g/L; (2) optimization of culture medium prescription.The patent mainly comprised has: CN101993824A, CN101831387B, CN101886044A, CN101914581B and CN103627640A, these patents are by changing the component in substratum, as prepared full-synthetic culture medium, optimize electrolytes and minerals content, reduce cl content and add external source fat compatible material as the mode such as trehalose, trimethyl-glycine, reach the object improving DHA output; (3) reduce production cost by optimization for fermentation technology mode, improve efficiency of pcr product.This type of patent comprises CN101519676B, CN101812484B, CN101824442B, CN102972533A, CN103146584A and CN103614427A.Comprehensive above-mentioned patent, the most high-biomass of schizochytrium limacinum reaches 125.2g/L, and DHA output is up to 33.5g/L.Therefore, obtaining the superior strain of DHA, and in conjunction with excellent zymotechnique, obtain higher biomass and fat content, is the key point of the industrialization promotion of DHA.
Summary of the invention
The object of this invention is to provide that a kind of fermentation period is short, fast growth the schizochytrium limacinum of high yield docosahexenoic acid and application thereof.
The technical solution used in the present invention is for achieving the above object:
A kind of schizochytrium limacinum, schizochytrium limacinum, its Classification And Nomenclature is schizochytrium limacinum (Aurantiochytriumsp) SD006, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on October 21st, 2013, its deposit number is CGMCCNo:8383.
Schizochytrium limacinum Aurantiochytriumsp.SD006 bacterial strain optimum temperuture is high, fast growth.Its optimum growth temperature is 28 DEG C-35 DEG C, ferments and can obtain maximum biomass and DHA output after 70 hours.
An application for schizochytrium limacinum, described schizochytrium limacinum is applied to be prepared in docosahexenoic acid.Specifically, with described schizochytrium limacinum for starting strain, high density fermentation obtains preparation DHA grease in the fermentation medium.
Described leavening temperature is 28 DEG C-35 DEG C, and fermentation time is 60-80 hour, in fermenting process, have employed and mends nitrogen operation, and in 24-48 hour interval of fermentation, stream adds the corn steep liquor solution of 50% (w/v).Have employed in whole fermenting process and mend sugar operation, in the hierarchy of control, glucose concn is between 20-60g/L, and during fermentation ends, glucose concn is not higher than 10g/L.
Described fermention medium is glucose 60-120g/L, yeast extract 5-30g/L, corn steep liquor 5-20g/L, potassium primary phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, Trisodium Citrate 0.5-5g/L, sea crystal 5-30g/L, vitamins B
130-200mg/L, vitamins B
630-200mg/L, vitamins B
125-50mg/L and vitamin H 2-50mg/L, is settled to 1L with water.
Prepare the method for producing docosahexenoic acid: with described schizochytrium limacinum for a starting strain, high density fermentation obtains and prepares docosahexenoic acid in the fermentation medium.
Described leavening temperature is 28 DEG C-35 DEG C, and fermentation time is 60-80 hour, in fermenting process, have employed and mends nitrogen operation, and in 24-48 hour interval of fermentation, stream adds the corn steep liquor solution of 50% (w/v).Have employed in whole fermenting process and mend sugar operation, in the hierarchy of control, glucose concn is between 20-60g/L, and during fermentation ends, glucose concn is not higher than 10g/L.
Described fermention medium is glucose 60-120g/L, yeast extract 5-30g/L, corn steep liquor 5-20g/L, potassium primary phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, Trisodium Citrate 0.5-5g/L, sea crystal 5-30g/L, vitamins B
130-200mg/L, vitamins B
630-200mg/L, vitamins B
125-50mg/L and vitamin H 2-50mg/L, is settled to 1L with water.
Compared with the prior art the present invention has following distinguishing feature and positively effect:
1. the present invention's screening obtains the schizochytrium limacinum bacterial strain of strain high yield DHA at relatively high temperatures, and this bacterial strain can grow fast under 28 DEG C of-35 DEG C of conditions, and accumulates docosahexenoic acid in a large number.
2. obtained strains fast growth of the present invention, fermentation time is short.Schizochytrium limacinum bacterial strain high density fermentation is utilized to produce docosahexenoic acid.Under above-mentioned fermentation condition, within 60-70 hour, maximum production can be obtained.After 70 hours fermentation, obtain biomass be 180-200g/L, DHA output is 50-65g/L.
3. obtained strains of the present invention screens from mangrove forest waters, Zhanjiang, Guangdong Province that obtain can the schizochytrium limacinum Aurantiochytriumsp.SD006 bacterial strain of high yield docosahexenoic acid, has good industrial application value.Screen can the schizochytrium limacinum of high yield DHA, and utilize the method for its high density fermentation production DHA grease.
Accompanying drawing explanation
The schizochytrium limacinum SD006 bacterial strain form under the microscope that Fig. 1 provides for the embodiment of the present invention, a is optical microphotograph Microscopic observation, and b is transmission electron microscope observing, and c is scanning electron microscopic observation.
The evolutionary tree of the schizochytrium limacinum SD006 bacterial strain 18SrRNA gene utilizing MEGA5 software to obtain that Fig. 2 provides for the embodiment of the present invention.
Embodiment
Embodiment 1
Schizochytrium limacinum, its Classification And Nomenclature is schizochytrium limacinum (Aurantiochytriumsp) SD006, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on October 21st, 2013, its deposit number is CGMCCNo:8383.
Described schizochytrium limacinum bacterial strain adopts Pollen Pini method of fishing to be separated from the rotted leaf of mangrove forest waters, Zhanjiang, Guangdong Province collection to obtain.Observe under an optical microscope: thalline is spheroidal or elliposoidal, diameter is at 10-20 micron, and the main divisional mode that adopts is bred.There are two flagellums (see Fig. 1) of the raw Length discrepancy in side.In born of the same parents, lipid acid composition is mainly tetradecanoic acid, hexadecanoic acid, clupanodonic acid (DPA) and docosahexenoic acid (DHA), and wherein DHA content accounts for more than 55% of total fatty acids.Compare to its 18SrDNA sequence with molecular biology method, find that the homology of the 18SrDNA sequence of this bacterial strain and schizochytrium limacinum is greater than 99%, therefore judge that this bacterial strain belongs to schizochytrium limacinum, this sequence is denoted as SEQIDNO.1 (see Fig. 2).
SEQIDNO.1
ccaacctggttgatcctgccagtagtcatatcctcgtctcaaagattaagccatgcatgt60
gtaagtataagcgattgtactgtgagactgcgaacggctcattatatcagtaataatttc120
ttcggtagtttcttttatatggatacctgcagtaattctggaaataatacatgctgtaag180
agccctgtatggggctgcacttattagattgaaggcgattttattggtgaatcatgataa240
ttgagcagattgactttttagtcgatgaatcgtttgagtttctgccccatcagttgtcga300
cggtagtgtattggactacggtgactataacgggtgacggagagttagggctcgactccg360
gagagggagcctgagagacggctaccatatccaaggatagcagcaggcgcgtaaattacc420
cactgtggactccacgaggtagtgacgagaaatatcgatgcgaagcgtgtatgtgttttg480
ctatcggaatgagagcaatgtaaaaccctcatcgaggatcaactggagggcaagtctggt540
gccagcagccgcggtaattccagctccagaagcatatgctaaagttgtcgcagttaaaaa600
gctcgtagttgaatttctggcatgggcgaccggtgctttccctgaatggggattgattgt660
ctgtgttgccttggccatctttttcttttctttattggggagaaatctttcactgtaatc720
aaagcagagtgttccaagcaggtcgtatgaccggtatgtttattatgggatgataagata780
ggacttgggtgctatttttgttggtttgcacgcctgagtaatggttaataggaacagttg840
ggggtattcgtatttaggagctagaggtgaaattcttggatttccgaaagacgaactaga900
gcgaaggcatttaccaagcatgttttcattaatcaagaacgaaagtctggggatcgaaga960
tgattagataccatcgtagtctagaccgtaaacgatgccgacttgcgattgttgggtgct1020
ttattaatgggcctcagcagcagcacatgagaaatcaaagtctttgggttccggggggag1080
tatggtcgcaaggctgaaacttaaaggaattgacggaagggcaccaccaggagtggagcc1140
tgcggcttaatttgactcaacacgggaaaacttaccaggtccagacataggtaggattga1200
cagattgagagctctttcatgattctatgggtggtggtgcatggccgttcttagttggtg1260
gagtgatttgtctggttaattccgttaacgaacgagacctcggcctactaaatagtgcgt1320
ggtatggcaacatagtgcgttttaacttcttagagggacatgtccggtttacgggcagga1380
agttcgaggcaataacaggtctgtgatgcccttagatgttctgggccgcacgcgcgctac1440
actgatgggttcatcgggttttaattctaatttttggaattgagtgcttggtcggaaggc1500
ctggctaatccttggaacgctcatcgtgctggggctagatttttgcgattattaatctcc1560
agcgaggaattcctagtaaacgcaagtcatcagcttgcattgaatacgtccctgcccttt1620
gtacacaccgcccgtcgcacctaccgattgaacggtccgatgaaaccatgggatgtttct1680
gtttggattcatttttggacagaggcagaactcgggtgaatcttattgtttagaggaagg1740
tgaagtcgtaacaagg1756
The available carbon source of schizochytrium limacinum Aurantiochytriumsp.SD006 bacterial strain comprises glucose, glycerine, fructose, molasses, starch saccharificating liquid and lignocellulose saccharified liquid.The suitableeest carbon source is glucose, and concentration is 60g/L-120g/L; To organic nitrogen source with inorganic nitrogen-sourcedly all can to utilize, wherein organic nitrogen source comprises yeast extract, peptone, Tryptones, corn steep liquor, urea; Inorganic nitrogen-sourcedly comprise inorganic ammonium salt and inorganic nitrate.The suitableeest nitrogenous source is yeast extract, and concentration is 10g/L-30g/L.In addition, corn steep liquor is also more suitable nitrogenous source, and concentration is 2g/L-10g/L.
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 30 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 30 DEG C, with the rotating speed of 200rpm, cultivates 12h, obtain secondary seed; 1.2L fermention medium is added, the secondary seed solution of access activation in 3L bio-reactor.In fermenting process, temperature controls at 28 DEG C.In fermenting process, have employed and mend nitrogen operation, in 24-48 hour of fermentation, stream adds the corn steep liquor solution of 50% (w/v).Automatic interpolation 2MNaOH or 14% citric acid make pH value remain on 6.5.Fermentation time 80 hours.
After fermentation ends, the output (see table 1) that 3L fermentor tank obtains schizochytrium limacinum is 180.43g/L, and obtain bio-oil 94.1g/L, DHA output is 53.5g/L.
Seed culture medium is glucose 60g/L, yeast extract 5g/L, corn steep liquor 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.5g/L, Trisodium Citrate 0.5g/L, sea crystal 5g/L, vitamins B
130mg/L, vitamins B
630mg/L, vitamins B
125mg/L and vitamin H 2mg/L.
Fermention medium is glucose 120g/L, yeast extract 30g/L, corn steep liquor 20g/L, potassium primary phosphate 8g/L, magnesium sulfate 5g/L, Trisodium Citrate 5g/L, sea crystal 30g/L, vitamins B
1200mg/L, vitamins B
6200mg/L, vitamins B
1250mg/L and vitamin H 50mg/L.
Lipid acid composition situation in schizochytrium limacinum SD006 born of the same parents after table 1 ferments
Embodiment 2
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 35 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 35 DEG C, with the rotating speed of 200rpm, cultivates 12h, obtain secondary seed; 1.2L fermention medium is added, the secondary seed solution of access activation in 3L bio-reactor.In fermenting process, temperature controls at 35 DEG C.In fermenting process, have employed and mend nitrogen operation, in 24-48 hour of fermentation, stream adds the corn steep liquor solution of 50% (w/v).Automatic interpolation 2MNaOH or 14% citric acid make pH value remain on 6.5.Fermentation time 60 hours.
After fermentation ends, the output that 3L fermentor tank obtains schizochytrium limacinum is 190.25g/L, and obtain bio-oil 104.6g/L, DHA output is 52.7g/L.
Above-mentioned, seed culture medium is glucose 60g/L, yeast extract 5g/L, corn steep liquor 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.5g/L, Trisodium Citrate 0.5g/L, sea crystal 5g/L, vitamin B13 0mg/L, vitamin B6 30mg/L, vitamin B12 5mg/L and vitamin H 2mg/L.
Fermention medium is glucose 60g/L, yeast extract 5g/L, corn steep liquor 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.5g/L, Trisodium Citrate 0.5g/L, sea crystal 5g/L, vitamins B
130mg/L, vitamins B
630mg/L, vitamins B
125mg/L and vitamin H 2mg/L.
Embodiment 3
The bacterial strain access being kept at glycerine pipe is equipped with in the 250ml shaking flask of 50ml seed culture medium, in the shaking table of 32 DEG C, with the rotating speed of 200rpm, cultivates 24h, obtain first order seed; First order seed access is equipped with in the 500ml shaking flask of 100ml seed culture medium, in the shaking table of 32 DEG C, with the rotating speed of 200rpm, cultivates 12h, obtain secondary seed; 3L fermention medium is added, the secondary seed solution of access activation in 5L bio-reactor.In fermenting process, temperature controls at 32 DEG C.In fermenting process, have employed and mend nitrogen operation, in 24-48 hour of fermentation, stream adds the corn steep liquor solution of 50% (w/v).Automatic interpolation 2MNaOH or 14% citric acid make pH value remain on 6.5.Fermentation time 70 hours.
After fermentation ends, the output that 5L fermentor tank obtains schizochytrium limacinum is 202.86g/L, and obtain bio-oil 114.1g/L, DHA output is 65.4g/L.
Above-mentioned, seed culture medium is glucose 60g/L, yeast extract 5g/L, corn steep liquor 5g/L, potassium primary phosphate 0.5g/L, magnesium sulfate 0.5g/L, Trisodium Citrate 0.5g/L, sea crystal 5g/L, vitamin B13 0mg/L, vitamin B6 30mg/L, vitamin B12 5mg/L and vitamin H 2mg/L.
Fermention medium is glucose 90g/L, yeast extract 15g/L, corn steep liquor 10g/L, potassium primary phosphate 5g/L, magnesium sulfate 2g/L, Trisodium Citrate 3g/L, sea crystal 15g/L, vitamins B
1100mg/L, vitamins B
6100mg/L, vitamins B
1220mg/L and vitamin H 20mg/L.
Claims (5)
1. a schizochytrium limacinum, it is characterized in that: schizochytrium limacinum, its Classification And Nomenclature is schizochytrium limacinum (Aurantiochytriumsp) SD006, be preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on October 21st, 2013, its deposit number is CGMCCNo:8383.
2. an application for schizochytrium limacinum according to claim 1, is characterized in that: described schizochytrium limacinum is applied to be prepared in docosahexenoic acid.
3. by the application of schizochytrium limacinum according to claim 2, it is characterized in that: with described schizochytrium limacinum for starting strain, high density fermentation obtains preparation DHA grease in the fermentation medium.
4. by the application of schizochytrium limacinum according to claim 3, it is characterized in that: described leavening temperature is 28 DEG C-35 DEG C, and fermentation time is 60-80 hour, in fermenting process, have employed and mend nitrogen operation, in 24-48 hour interval of fermentation, stream adds the corn steep liquor solution of 50% (w/v).
5. by the application of schizochytrium limacinum according to claim 3, it is characterized in that: described fermention medium is glucose 60-120g/L, yeast extract 5-30g/L, corn steep liquor 5-20g/L, potassium primary phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, Trisodium Citrate 0.5-5g/L, sea crystal 5-30g/L, vitamins B
130-200mg/L, vitamins B
630-200mg/L, vitamins B
125-50mg/L and vitamin H 2-50mg/L.
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| CN105420122A (en) * | 2015-12-23 | 2016-03-23 | 通威股份有限公司 | Schizochytrium limacinum suitable for high-density culture and method for producing grease rich in DHA |
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| CN117844646B (en) * | 2023-12-14 | 2024-05-24 | 陕西海斯夫生物工程有限公司 | Schizochytrium limacinum with high DHA yield and application thereof |
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