CN104560731B - A kind of yeast-like fungi of high yield squalene and its application - Google Patents
A kind of yeast-like fungi of high yield squalene and its application Download PDFInfo
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Abstract
The present invention relates to the application technologies of marine microorganism, and in particular to a kind of ocean yeast-like fungi SD301 bacterial strains and the method using its high density fermentation production squalene.Yeast-like fungi Classification And Nomenclature is yeast-like fungi (Pseudozyma sp.), has been preserved in the China Committee for Culture Collection of Microorganisms's common micro-organisms center positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and deposit number is CGMCC No：9687, the deposit date is Septembers in 2014 19 days.Application of the yeast-like fungi in fermenting and producing squalene.The squalene purity that method using the present invention obtains reaches 90% or more, and whole set process is easy to operate, reduces cost, is suitble to industrialized production.
Description
Technical field
The present invention relates to the application technologies of marine microorganism, and in particular to a kind of ocean yeast-like fungi SD301 bacterial strains and profit
The method for producing squalene with its high density fermentation.
Background technology
Squalene (squalene) is a kind of lipid unsaponifiable matter, and its chemical name is 2,6,10,15,19,23- pregnancy
Base -2,6,10,14,18,22- tetracosa carbon, six alkene belong to open chain triterpene, have the internal superoxide dismutase (SOD) of raising living
Property, enhancing body immunity, improve sexual function, anti-aging, the different physiological roles such as antifatigue, antitumor, be a kind of nontoxic
Property there is the marine biomaterial of effect of preventing and curing diseases, and be widely used in medicine, beauty, cosmetics, food etc. respectively
A field.The source of squalene is mainly fish oil at present, especially dogfish oil.But with the increasingly depleted and phase of fishery resources
Becoming better and approaching perfection day by day for regulation is closed, promotes people to strive to find other squalene sources, such as from olive oil, palm oil, other oils
Crop, or separation is extracted from Amaranthus, seed, rice bran, wheat embryo, but squalene content pole in plant
Low, about accounting is 0.1 to 0.7% by weight.
Another alternative solution is to produce squalene using the method for microbial fermentation.Such as patent CN 102787074
A, 103748212 A of CN disclose one plant production squalene yeast-like fungi strain its dry mycelium in squalene mass percentage
It is 1%；102666837 A of patent CN produce squalene mass content using Yarrowia lipolytica and account for intracellular total lipid
2%；And 103266137 B of patent CN then utilize transgenic escherichia coli to produce squalene.But the defect that these patents are common
Be squalene in unit fermentation volume yield all be far below 1g/L.In document report, spiny dogfish is produced using microbial fermentation
The yield of alkene is then lower, if 6063 bacterial strain squalene yield of thraustochytriale ACEM is about 0.1 milligrams per gram biomass (referring to Le
Tie up this et al.《Neoformation technology》, 2001, the 439-447 pages.), and Schizochytrium mangrovei FB1 bacterial strains
Yield be about 0.162 milligrams per gram biomass (referring to：River et al.,《Agricultural and Food Chemistry magazine》, 2004, the 52nd
Phase, number of pages 1196 to 1200), and the yield of Aurantiochytrium BR-MP4-A1 bacterial strains is about the life of 0.18 milligrams per gram
Object amount (referring to：Lee et al.,《Agricultural and Food Chemistry magazine》, 2009, the 57th phase, number of pages 4267 to 4272), and often et al.
It produces the ability highest of squalene to 207 bacterial strains of yeast-like fungi Pseudozyma sp.JCC being separated to, and only 340.52 in the least
Grams per liter zymotic fluid.Therefore, the microorganism for screening high squalene yield is to solve current biological fermentation process production squalene industry to close
The effective means of key bottleneck.
Invention content
The object of the present invention is to provide a kind of yeast-like fungi of high yield squalene and its applications.
To achieve the above object, the invention adopts a technical scheme as：
A kind of yeast-like fungi of high yield squalene, yeast-like fungi Classification And Nomenclature are yeast-like fungi (Pseudozyma sp.),
The China Committee for Culture Collection of Microorganisms being preserved in positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 is commonly micro-
Bio-Centers, deposit number are CGMCC No：9687, the deposit date is Septembers in 2014 19 days.
A kind of application of the yeast-like fungi of high yield squalene, application of the yeast-like fungi in fermenting and producing squalene.
A kind of method of high density fermentation production squalene, with CGMCC No：9687 bacterial strains are starting strain, are trained in liquid
It supports base middle-high density to ferment, isolated somatic cells, by somatic cells through broken, extraction, refined acquisition squalene.
Specially：
It is inoculated in seed culture medium 1. the strain that will be stored in the glycerine tube, inoculum concentration 3-5% (v/v), at 20-30 DEG C,
With the rotating speed of 150-200rpm, 24-48h is cultivated, obtains first order seed；
2. first order seed is accessed in seed culture medium, inoculum concentration 3-5% (v/v), at 20-30 DEG C, with 150-200rpm
Rotating speed, cultivate 24-48h, obtain secondary seed；
3. secondary seed solution is accessed in fermentation medium, inoculum concentration 2-10% (v/v), ventilatory capacity 0.2-2vvm, stirring
Rotating speed 200-800rpm, 15-37 DEG C of tank temperature, pH 4-9, ferment 60-120h, fermenting and producing squalene.
It is 150-180g/L that biomass is obtained after above-mentioned fermentation, and squalene yield is 9-15g/L.
In the seed culture medium, carbon source content is 30-60g/L, and nitrogen source content is 10-20g/L, solvent 1:1 (quality
Than) seawater and distilled water mixture.
The carbon source is glucose, glycerine, fructose, xylose, sucrose, maltose, molasses, starch saccharificating liquid or wood fibre
Plain saccharified liquid；The nitrogen source is organic nitrogen source or inorganic nitrogen-sourced.
The organic nitrogen source is yeast extract, peptone, tryptone, corn steep liquor, beef extract, soybean protein, paddy
Propylhomoserin sodium or urea, it is described inorganic nitrogen-sourced for ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, ammonium hydroxide or potassium nitrate.
It include following component in the fermentation medium：Glucose 20-120g/L, yeast extract 0.2-30g/L, albumen
Peptone 0.2-20g/L, potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 5-30g/L, vitamin B1 3-200mg/
L, vitamin B6 3-200mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
The mode of the secondary seed fermented and cultured is batch fermentation, fed-batch fermentation, continuously ferments or semicontinuous hair
Ferment.
The separation method is centrifugation, filtering or flocculation；The breaking method is extrusion wall-breaking or enzymatic isolation method broken wall；It is described
Extraction, purification step are to recycle the crude oil containing squalene using nonpolar solvent, and the crude oil molecular distillation is obtained squalene.
The nonpolar solvent is the mixed solvent of n-hexane or n-hexane-ethyl alcohol；The molecular distillation be 190~
Main distillate fraction squalene is steamed under the conditions of 210 DEG C and 5~20Pa of pressure.
It, by centrifuge (centrifugal force 4500g) separation and fermentation liquid, is 65 by mass ratio to be further:1 wet thallus
It is mixed with glusulase and carries out broken wall, add the n-hexane isometric with wet thallus and isometric ethyl alcohol later, be uniformly mixed
Stand 4 hours afterwards.Upper organic phase is recycled into organic solvent, residue obtains squalene crude product.
Compared with the prior art the present invention has following distinguishing feature and good effect：
(1) it screens to obtain the yeast-like fungi bacterial strain of a high yield of squalene, the bacterium from Zhanjiang, Guangdong Province mangrove waters
Strain can under the conditions of 15 DEG C -37 DEG C fast-growth, and largely accumulate squalene.
(2) speed of growth is fast, and fermentation time is short, and squalene yield is big.It under the above fermentation conditions, 80-100 hours can
Maximum production is obtained, acquisition biomass is 150-180g/L, and squalene yield is 9-15g/L.
(3) product purity is high.In the above extraction and molecular distillation conditions, the squalene purity of acquisition is more than 90%.
Description of the drawings
Fig. 1 is the form of yeast-like fungi SD301 bacterial strains provided in an embodiment of the present invention under the microscope, and a is optical microphotograph
Under the microscope, b is scanning electron microscopic observation；
Fig. 2 is the gas chromatogram of yeast-like fungi SD301 bacterial strains squalene content analysis provided in an embodiment of the present invention；
Fig. 3 is the yeast-like fungi SD301 bacterial strain ITS sequences provided in an embodiment of the present invention obtained using MEGA5 softwares
Chadogram.
Specific implementation mode
One plant of gained of the invention can high yield squalene 301 bacterial strains of yeast-like fungi Pseudozyma sp.SD, have fine
Industrial application value.
Embodiment 1：Utilization of the yeast-like fungi to carbon source
Yeast-like fungi is screened from the mangrove waters of Zhanjiang, Guangdong Province and obtains, and Classification And Nomenclature is yeast-like fungi
(Pseudozyma sp.), laboratory are named as yeast-like fungi SD301 bacterial strains, have been preserved in positioned at Chaoyang District, Beijing City North Star west
The China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) of the institute 3 of road 1, deposit number are
CGMCC No：9687, preservation date September in 2014 19 days.
The yeast-like fungi SD301 bacterial strains are cylindrical yeast shape vegetal cells, and thalline is about 3-6um, fusiform spore
Son is being grown in thalline one end (Fig. 1).There is the accumulation of a large amount of squalenes, squalene content to account for the quality of dry cell weight into the cell for it
Than being more than 5% (Fig. 2).(Fig. 3) is compared to its ITS regional sequence with molecular biology method, find the bacterial strain with
The homology for the ITS sequence that Pseudozyma belongs to is more than 99%, therefore, it is determined that this bacterial strain belongs to yeast-like fungi, which is denoted as
SEQ ID NO.1。
Utilization of the yeast-like fungi to carbon source
In the triangle shake bottle of 250mL, 50mL culture mediums are configured, nitrogen source is yeast extract 20g/L, and salinity 15 is divided
Different carbon sources (glucose, glycerine, fructose, xylose, sucrose, maltose, molasses, starch saccharificating liquid or wood fibre are not added
Plain saccharified liquid), a concentration of 60g/L, it is 6.0 to adjust pH value, after high pressure sterilization, the strain seed liquor of 5mL precultures is accessed, in air
28 DEG C of shaken cultivations 5 days on oscillator are bathed, oscillation rotating speed is 180rpm.Thalline were collected by centrifugation, is freeze-dried to constant weight, and it is dry to survey it
Weight；Part thalline is taken, the percentage composition of squalene in thalline is measured by GC-MS.It the results are shown in Table 1.
Utilization of the 1 yeast-like fungi SD301 bacterial strains of table to carbon source
Utilization of the yeast-like fungi to nitrogen source
In the triangle shake bottle of 250mL, configuration 50m culture mediums use glucose for carbon source, concentration 60g/L, and salinity is
15, be separately added into a concentration of 20g/L organic nitrogen source (yeast extract, peptone, tryptone, corn steep liquor, beef extract,
Soybean protein, sodium glutamate or urea) and 5g/L inorganic nitrogen-sourced (ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, ammonium hydroxide or nitre
Sour potassium), it is 6.0 to adjust pH value, after high pressure sterilization, the strain seed liquor of 5mL precultures is accessed, 28 DEG C on air bath oscillator
Shaken cultivation 5 days, oscillation rotating speed are 180rpm.Analysis method is same as above, and experimental result is shown in Table 2.
Utilization of the 2 yeast-like fungi SD301 bacterial strains of table to nitrogen source
2 3L fermentation tanks of embodiment are tested
In 250mL shaking flasks of the access equipped with 50mL seed culture mediums that the strain that will be stored in the glycerine tube, in 20 DEG C of shaking table
In, with the rotating speed of 150rpm, 48h is cultivated, obtains first order seed；By first order seed access equipped with 100mL seed culture mediums
In 500mL shaking flasks, in 20 DEG C of shaking table, with the rotating speed of 150rpm, 48h is cultivated, obtains secondary seed；To 3L bioreactors
Middle addition 1.2L fermentation mediums, access the secondary seed solution of activation.In fermentation process, ventilatory capacity 0.2vvm, speed of agitator
200rpm, 15 DEG C, pH 4 of tank temperature, fermentation time 120 hours.Every time when concentration of glucose is down to 10g/L or so, flow feeding
Liquid makes sugared concentration be restored to 60g/L or so.In fermentation process, feed supplement 4 times altogether.
Wherein, in seed culture medium, glucose 30g/L, yeast powder content is 10g/L, solvent 1:1 (mass ratio)
The mixture of seawater and distilled water.
It include following component in fermentation medium：Glucose 120g/L, yeast extract 30g/L, peptone 0.2g/L, phosphorus
Acid dihydride potassium 8g/L, magnesium sulfate 5g/L, sea crystal 30g/L, vitamin B1 200mg/L, vitamin B6 200mg/L, vitamin
B12 50mg/L, biotin 50mg/L.
Feed supplement liquid group is divided into：Glucose 800g/L, yeast extract 50g/L.
Biomass calculates：
It takes zymotic fluid 30mL, 4000g to centrifuge 10min, collects thalline, weigh its dry mycelium quality after dry, and be converted to
Biological yield, in terms of gram per liter zymotic fluid (g/L).
The preparation of squalene
By centrifuge (centrifugal force 4500g) separation and fermentation liquid, wet thallus is collected, is 65 by mass ratio:1 wet thallus with
Glusulase mixing carries out broken wall, adds the n-hexane isometric with wet thallus and isometric ethyl alcohol later, after mixing
Stand 4 hours.Upper organic phase is recycled into organic solvent, residue obtains squalene crude product.
Squalene detection method
Using Agilent 7890B gas-chromatographies, column model is HP-5 columns (30m × 0.25mm × 0.25 μm)；It carries
Gas is high pure nitrogen, constant current mode, flow velocity 1.0mL/min；Sampling volume is 1 μ L, and detector is hydrogen ion flame detector.It rises
Warm program is：100 DEG C of holding 1min of initial temperature, rise to 300 DEG C with 15 DEG C/min, keep 6min.After squalene standard test, should
Under the conditions of the chromatographic peak that occurs of 12.86min or so be spiny dogfish olefinic substance peak.
After fermentation, the yield that 3L fermentation tanks obtain class yeast is 162.39g/L, and squalene yield is 11.69g/L.
3 10L fermentation tanks of embodiment are tested
In 250mL shaking flasks of the access equipped with 50mL seed culture mediums that the strain that will be stored in the glycerine tube, in 30 DEG C of shaking table
In, with the rotating speed of 200rpm, culture for 24 hours, obtains first order seed；By first order seed access equipped with 100mL seed culture mediums
In 500mL shaking flasks, in 30 DEG C of shaking table, with the rotating speed of 200rpm, culture for 24 hours, obtains secondary seed；To 10L biological respinses
6L fermentation mediums are added in device, access the secondary seed solution of activation.In fermentation process, ventilatory capacity 2vvm, speed of agitator
800rpm, 37 DEG C, pH 9 of tank temperature, fermentation time 60 hours.Every time when concentration of glucose is down to 10g/L or so, flow feeding
Liquid makes sugared concentration be restored to 60g/L or so.In fermentation process, feed supplement 4 times altogether.
Wherein, in seed culture medium, glucose 60g/L, yeast powder content is 20g/L, solvent 1:1 (mass ratio)
The mixture of seawater and distilled water.
It include following component in fermentation medium：Glucose 20g/L, yeast extract 0.2g/L, peptone 20g/L, phosphorus
Acid dihydride potassium 0.5g/L, magnesium sulfate 0.5g/L, sea crystal 5g/L, vitamin B1 3mg/L, vitamin B6 3mg/L, vitamin
B12 1mg/L, biotin 1mg/L.
Feed supplement liquid group is divided into：Glucose 800g/L, yeast extract 50g/L.
After fermentation, the yield that 10L fermentation tanks obtain class yeast is 150.43g/L, and squalene yield is 9.03g/L.
4 100L fermentation tanks of embodiment are tested
In 250mL shaking flasks of the access equipped with 50mL seed culture mediums that the strain that will be stored in the glycerine tube, in 28 DEG C of shaking table
In, with the rotating speed of 200rpm, culture for 24 hours, obtains first order seed；By first order seed access equipped with 100mL seed culture mediums
In 500mL shaking flasks, in 28 DEG C of shaking table, with the rotating speed of 200rpm, culture for 24 hours, obtains secondary seed；To 100L biological respinses
65L fermentation mediums are added in device, access the secondary seed solution of activation.In fermentation process, ventilatory capacity 1.7vvm, speed of agitator
600rpm, 28 DEG C, pH7 of tank temperature, fermentation time 120 hours.Every time when concentration of glucose is down to 10g/L or so, flow feeding
Liquid makes sugared concentration be restored to 60g/L or so.In fermentation process, feed supplement 5 times altogether.
Wherein, in seed culture medium, glucose 60g/L, yeast powder content is 20g/L, solvent 1:1 (mass ratio)
The mixture of seawater and distilled water.
It include following component in fermentation medium：Glucose 120g/L, yeast extract 30g/L, peptone 10g/L, phosphorus
Acid dihydride potassium 8g/L, magnesium sulfate 5g/L, sea crystal 30g/L, vitamin B1 200mg/L, vitamin B6 200mg/L, vitamin
B12 50mg/L, biotin 50mg/L.
Feed supplement liquid group is divided into：Glucose 800g/L, yeast extract 50g/L.
After fermentation, the yield that 100L fermentation tanks obtain class yeast is 182.25g/L, and squalene yield is 15.49g/
L。
The preparation of 5 squalene fine work of embodiment
The zymotic fluid that 2.5 tons of above-described embodiments obtain is detached by disc centrifuge (centrifugal force 4500g), is collected altogether wet
1 ton of thalline, the glusulase that 1.5% weight of wet thallus quality is added carry out broken wall, add 1 ton of n-hexane later, and 1 ton of ethyl alcohol is mixed
4 hours are stood after closing uniformly.Upper organic phase evaporation recycling organic solvent is taken, squalene crude product is obtained.Crude product is transferred to steaming
In distillation unit, at 190 DEG C, pressure is distilled to obtain squalene fine work 26.8Kg under the conditions of being 5Pa, and purity is more than after testing
93%.
The preparation of 6 squalene fine work of embodiment
The zymotic fluid that 10 tons of above-described embodiments obtain is detached by disc centrifuge (centrifugal force 4500g), is collected altogether wet
About 4.3 tons of thalline, the glusulase that 1.5% weight of wet thallus quality is added carry out broken wall, add 4 tons of n-hexanes later, mix
4 hours are stood after uniformly.Upper organic phase evaporation recycling organic solvent is taken, squalene crude product is obtained.Crude product is transferred to distillation
In device, at 210 DEG C, pressure is distilled to obtain squalene fine work about 128Kg under the conditions of being 20Pa, and purity is more than after testing
90%.
Claims (6)
1. a kind of yeast-like fungi of high yield squalene, it is characterised in that：Yeast-like fungi Classification And Nomenclature is yeast-like fungi
(Pseudozyma sp.) has been preserved in the Chinese microorganism strain preservation positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Administration committee's common micro-organisms center, deposit number are CGMCC No：9687, the deposit date is Septembers in 2014 19 days；Institute
It is SEQ ID NO.1 to state yeast-like fungi ITS regional sequences.
2. a kind of application of the yeast-like fungi of high yield squalene described in claim 1, it is characterised in that：The yeast-like fungi exists
Application in fermenting and producing squalene.
3. a kind of method of high density fermentation production squalene, it is characterised in that：With CGMCC No described in claim 1：
9687 bacterial strains are starting strain, in liquid medium high density fermentation, isolated somatic cells, by somatic cells through broken
Broken, extraction, refined acquisition squalene.
4. the method for high density fermentation production squalene as described in claim 3, it is characterised in that：
It is inoculated in seed culture medium 1. the strain that will be stored in the glycerine tube, inoculum concentration 3-5%v/v, at 20-30 DEG C, with 150-
The rotating speed of 200rpm cultivates 24-48h, obtains first order seed；
2. first order seed is accessed in seed culture medium, inoculum concentration 3-5%v/v, at 20-30 DEG C, with the rotating speed of 150-200rpm,
24-48h is cultivated, secondary seed is obtained；
3. secondary seed solution is accessed in fermentation medium, inoculum concentration 2-10%v/v, ventilatory capacity 0.2-2vvm, speed of agitator
200-800rpm, 15-37 DEG C of tank temperature, pH 4-9, ferment 60-120h, fermenting and producing squalene；
It include following component in the fermentation medium：Glucose 20-120g/L, yeast extract 0.2-30g/L, peptone
0.2-20g/L, potassium dihydrogen phosphate 0.5-8g/L, magnesium sulfate 0.5-5g/L, sea crystal 5-30g/L, vitamin B1 3-200mg/L,
Vitamin B6 3-200mg/L, vitamin B12 1-50mg/L, biotin 1-50mg/L.
5. the method for high density fermentation production squalene as described in claim 4, it is characterised in that：The seed culture medium
In, carbon source content is 30-60g/L, and nitrogen source content is 10-20g/L, and solvent is mass ratio 1:The mixing of 1 seawater and distilled water
Object.
6. the method for high density fermentation production squalene as described in claim 5, it is characterised in that：The carbon source is grape
Sugar, glycerine, fructose, xylose, sucrose, maltose, molasses, starch saccharificating liquid or lignocellulosic saccharified liquid；The nitrogen source is to have
Machine nitrogen source is inorganic nitrogen-sourced.
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| CN105920580B (en) * | 2016-04-28 | 2019-04-19 | 中山大学 | Ocean microorganism compound P-3 is preparing the application in antiaging agent |
| CN107574185A (en) * | 2017-09-11 | 2018-01-12 | 天津大学 | The thraustochytriale fermentation medium and its cultural method of a kind of high yield squalene |
| CN108866112B (en) * | 2018-08-17 | 2021-04-27 | 青岛中科潮生生物技术有限公司 | Method for preparing squalene by adopting lignocellulose |
| CN114854608B (en) * | 2022-04-18 | 2024-05-07 | 自然资源部第三海洋研究所 | Degradable polyurethane yeast fungus strain, identification method and application |
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